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Lectin-Microorganism

Interactions

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Interactions

c&©Jo~c&©J [b)w

[R1 o Jl [Q)©W~® University of Louisville

Louisville, Kentucky

Allegheny General Hospital and Medical College of Pennsylvania

Pittsburgh, Pennsylvania

C\ Taylor & Francis ~ Taylor&FrancisGroup

LONDON AND NEW YORK

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Library of Congress Cataloging-in-Publication Data

Lectin-microorganism interactions/edited by R. J. Doyle, Malcolm Slifkin.

p. em. Includes bibliographical references and index. ISBN 0-8247-9113-4 (alk. paper) 1. Lectins. 2. Microbial polysaccharides. I. Doyle, Ronald J.

II. Slifkin, Malcolm. [DNLM: 1. Lectins--physiology. 2. Lectins--diagnostic use.

3. Microbiology.] QP552.L42L415 1994 574.19'245--dc20 93-46008

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Preface

Lectins have become irreplaceable tools for modern microbiologists, molec-ular biologists, and biochemists. These carbohydrate-binding proteins have been employed as diagnostic reagents for viruses, bacteria, fungi, and pro-tozoa. They have also been used in epidemiological investigations in in-fectious diseases. The use of lectins to isolate microbial toxins, microbial mutants, cell surface glycoconjugates, and viral coat glycoproteins is now well established. This is the first book devoted solely to lectin-microorgan-ism interactions. The text contains an introduction to lectins and their interac-tions with microorganisms as well as chapters on lectins as probes for viral, bacterial, fungal and protozoal surfaces. Two chapters were contributed by Russian scientists who have reviewed much of the lectin-microorganism liter-ature from Eastern Europe. Another chapter is concerned about advances in the use of lectins in studying blood groups. Although blood cells do not qual-ify as microorganisms, the traditional association between blood bank labo-ratories and diagnostic microbiology laboratories is strong enough to justify a chapter on lectin-red cell interactions. A proposal to provide a uniform meth-od to abbreviate lectins is also presented. The book's abundant references en-compass most of the literature on lectin interactions with microorganisms. The text provides a thorough overview of lectin-microorganism interactions including applications and fundamental aspects ofthe interactions.

A list of the most common applications of lectins in microbiology and serology would include the following:

Diagnostic microbiology Epidemiological characterization of microorganisms

iii

iv

Research on protozoa Applications to fungi and yeasts Routine grouping of erythrocytes Automated blood-grouping apparatuses Assembly of cell surface in Bacillus subtilis Study of bacteriophage receptors Use in microbial ultrastructure Purification of teichoic acids Purification of viral components Characterization of glycoproteins in tissue culture cells Purification of microbial enzymes important in biotechnology Mechanism of bacterial adhesion Solution structure of teichoic acids Mechanism of root nodulation Structural determination of microbial polysaccharides

Preface

Characterization of lipopolysaccharide structure of Neisseria gonorrhoeae

This book is intended for all scientists who employ lectins as tools. Although the book does not provide detailed methods or physicochemical descriptions of lectins, microbiologists, biochemists, bio-tech engineers, physicians, epidemiologists, serologists, and other health care workers will find this volume an invaluable resource. The book can also serve as a text for a one-semester course in lectins and their applications to microbiology.

The versatility of lectins as reagents and tools in microbiology and serology is emphasized throughout the book. The availability of new lectins with new specificities will make it possible to identify even more applica-tions for lectins in microbiology and serology.

R. J. Doyle Malcolm Slifkin

Contents

Preface Contributors

1. Introduction to Lectins and Their Interactions

iii vii

with Microorganisms 1 R. J. Doyle

2. Use of Lectins in General and Diagnostic Virology 67 Sigvard Olofsson, Stig Jeansson, and John-Erik Stig Hansen

3. Epidemiological Applications of Lectins to Agents of Sexually Transmitted Diseases 111 William 0. Schalla and Stephen A. Morse

4. Application of Lectins in Clinical Bacteriology 143 Malcolm Slifkin

5. Lectin Specificities Relevant to the Medically Important Yeast Candida albicans 173 Hans C. Korting and Markus W. 01/ert

6. Lectin-Leishmania Interaction 191 R. L. Jacobson

7. Trypanosome-Lectin Interactions 225 Justus Schotte/ius and Martins S. 0. Aisien

v

vi Contents

8. Lectin Sorbents in Microbiology 249 V. M. Lakhtin

9. Microbial Lectins for the Investigation of Glycoconjugates 299 K. L. Shakhanina, N. L. Kalinin, and V. M. Lakhtin

10. Lectin-Blood Group Interactions 327 C. Levene, Nechama Gilboa-Garber, and Nachman C. Garber

Index 393

Contributors

Martins S. 0. Aisien Department of Zoology, University of Benin, Benin City, Nigeria

R. J. Doyle Professor of Microbiology, Department of Microbiology, School of Medicine; Associate Dean for Research, School of Dentistry, University of Louisville, Louisville, Kentucky

Nachman C. Garber Professor of Microbiology, Department of Life Sci-ences, Bar-Han University, Ramat-Gan, Israel

Nechama Gilboa-Garber Professor of Biochemistry, Department of Life Sciences, Bar-Han University, Ramat-Gan, Israel

John-Erik Stig Hansen Head of Research, Laboratory of Infectious Dis-eases, Hvidovre Hospital, Hvidovre, Denmark

R. L. Jacobson Medical Parasitologist, Department of Parasitology, He-brew University-Hadassah Medical School, Jerusalem, Israel

Stig Jeansson Associate Professor, Department of Clinical Virology, Uni-versity of Gothenburg, Gothenburg, Sweden

N. L. Kalinin Department of Biological Sciences (Immunology), Gamaleya Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow, Russia

vii

viii Contributors

Hans C. Korting Department of Dermatology, University of Munich, Mu-nich, Germany

V. M. Lakhtin Head, Laboratory' of Lectinology, Institute for Applied Sci-ence of Moscow University, and Institute of Food Substances, Russian Academy of Medical Sciences, Moscow, Russia

C. Levene Director, Reference Laboratory for Immunohematology and Blood Groups, Ministry of Health, Jerusalem, Israel

Stephen A. Morse Director, Division of Sexually Transmitted Diseases Lab-oratory Research, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Atlanta, Georgia

Markus W. Ollert Department of Biochemistry and Molecular Biology, University of Hamburg, Hamburg, Germany

Sigvard Olofsson Associate Professor, Department of Clinical Virology, University of Gothenburg, Gothenburg, Sweden

William 0. Schalla Chief, Model Performance Evaluation Program, Divi-sion of Laboratory Systems, Centers for Disease Control and Prevention, Public Health Service, U.S. Department of Health and Human Services, Atlanta, Georgia

Justus Schottelius Privatdozent, Department of Protozoology, Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany

K. L. Shakhanina Head, Department of Biological Sciences (Immunology), Gamaleya Institute of Epidemiology and Microbiology, Russian Academy of Medical Sciences, Moscow, Russia

Malcolm Slifkin Head, Section of Microbiology, Department of Labora-tory Medicine, Allegheny General Hospital; Professor of Microbiology and Immunology, and Professor of Pathology and Laboratory Medicine, Medi-cal College of Pennsylvania, Allegheny Campus, Pittsburgh, Pennsylvania


Interactions

1 Introduction to Lectins and Their Interactions with Microorganisms

R. ). DOYLE University of Louisville, Louisville, Kentucky

I. INTRODUCTION AND THE DEFINITION OF A LECTIN

Lectin research is now more than 100 years old. Most lectinologists ac-knowledge the valuable contribution of Stillmark [1] as the beginning of the centennial on lectin identification, purification, characterization, biological properties, and functions. Stillmark, for his Ph.D. thesis at the University of Dorpat (now Tartu, in Estonia), recorded the hemagglutinating proper-ties of extracts of Ricinus communis seeds and of members of the family Euphorbaceae. He observed that red cells of some species were refractory to hemagglutination, giving rise to the concept of lectin specificity. Since the 1960s, lectin research has seemed to gain an exponential strength. This has necessitated a critical examination of the word "lectin," followed by new attempts to define a lectin.

The original definition of a lectin was proposed by Boyd and Shapleigh to account for the blood group specificity of plant extracts. The word lectin itself is taken from the Latin Iegere, meaning to select or choose. As a tangential comment, Boyd and Shapleigh [2] made the prophetic statement "They [the lectins] promise to have practical and theoretical importance." Some researchers simply called the extracts possessing blood group specific-ity as agglutinins, hemagglutinins, or phytohemagglutinins. As pointed out by Boyd and Shapleigh [2], some immunologists did not seem very happy by applying the word "agglutinin" to a material from a plant tissue. The word lectin is now in much more common use, no doubt because red cell-agglutinating substances are found in almost all living tissues examined.

Because of the genesis of lectin research, Goldstein et al. [3] were

1

2 Doyle

prompted to redefine a lectin. They proposed a lectin as "a sugar-binding protein or glycoprotein of non-immune origin which agglutinates cells and or precipitates glycoconjugates." This definition assumes that alllectins are multivalent. It assumes that the specificity of the lectin is largely dependent on monosaccharide terminii. The definition takes into account the fact that lectins may be soluble or tissue-bound. The definition ascribes lectinlike properties to certain enzymes, such as amylases or phosphorylases, which may precipitate polysaccharides. Kocourek and Horejsi [4] contested the definition of lectin of Goldstein et al. [3]. They proposed that "lectins are sugar-binding proteins or glycoproteins of non-immune origin which are devoid of enzymatic activity towards sugars to which they bind and do not require free glycosidic hydroxyl groups on these sugars for their binding." This definition, therefore, dispenses with enzymes as lectins, and it also dispenses with the requirement of multivalency. Kocourek and Horejsi [4] agree that lectins are nonimmune proteins. Dixon [5], on behalf of the Nomenclature Committee of the International Union of Biochemistry, ac-cepted the definition of Goldstein et al. [3] for a lectin. Dixon argued that the definition proposed by Kocourek and Horejsi [4] was" ... too broad to be useful, since it includes substances such as sugar-transport proteins, chemotaxis receptors, certain bacterial toxins, hormones and interferons." Dixon further argued that some " . . . certain proteins hitherto known as lectins possess glycosidase activity." Dixon and the committee chose to remove the word "glycoprotein" from the Goldstein et al. definition because glycoproteins are a class of proteins. It seems clear that a single, simple definition of a lectin may be impossible. Both the Goldstein et al. and the Kocourek and Horejsi definitions have merit and the criticisms of Dixon are reasonable. The now-known multiple functions of lectins may compro-mise any of the foregoing definitions. Barondes [6] has now made a con-vincing attempt to establish a new definition for lectins. Barondes has pointed out that many of the well-characterized lectins have binding sites for noncarbohydrate ligands. For example, discoidin I, a multivalent pro-tein from Dictyostelium discoideum, binds N-acetylgalactosamine (Gal-NAc) and galactose (Gal) and contains an Arg-Gly-Asp (RGD) sequence. The RGD sequence is important to cell-substratum adhesive events in ani-mal cells, but it is also required for the developmental cycle of D. dis-coideum. Small peptides containing the RGD sequence interfere with devel-opmental processes of the slime mold. Therefore, it is clear that discoidin I is bifunctional, of which one function or property is dependent on carbohy-drate and the other on a noncarbohydrate-binding amino acid sequence. Similarly, the asialoglycoprotein receptor (Gal,-GalNAc-specific lectin) also contains an amino acid sequence that tethers it to cellular membranes. Barondes [6], in an effort to take into account the known properties of

Lectin-Microorganism Complexes 3

carbohydrate-binding proteins, has defined a lectin as "a carbohydrate-binding protein other than an enzyme or an antibody." This is the most satisfying, least-restrictive definition of a lectin yet proposed. But this definition is not perfect, as it must include periplasmic (nonmembrane-anchored) carbohydrate transport proteins of bacteria, such as the arabin-ose- and galactose-binding proteins of Escherichia coli. It also may be that certain proteins, such as limulin, a sialic acid-binding protein from the horseshoe crab Limulus polyphemus is a type of "immune" protein, exhibit-ing antibodylike properties. Nevertheless, the definition of a lectin given by Barondes will be adhered to in this book. A lucid and detailed history of the development of research on lectins has been given by Kocourek [7].

This book is concerned with the interactions between lectins and micro-organisms, including bacteria, fungi and yeasts, protozoa, metazoa, and viruses. Only a brief review will be presented on the properties of lectins. Lectins of microorganisms will not be discussed in terms of their functional roles as adhesins. A comprehensive review of the chemical and biological properties of lectins and their functions and applications was published in 1986 [8]. A readable account of lectinology has also been published by Sharon and Lis [9]. No single comprehensive review on lectin-microorgan-ism interactions is available, although reviews by Pistole [10], Doyle and Keller [11], Slifkin and Doyle [12] and Doyle and Slifkin [13] outline se-lected areas of the lectin-microbe literature. Table 1 provides a brief de-scription of selected papers on lectins and lectin-microorganism complexes. The table is designed to provide an overview of how lectins have been used to study microbial surfaces and glycoconjugates. Some of the experiments cited in Table 1 will be discussed more thoroughly in this and other chapters of the book.

II. SOURCES AND FUNCTION OF LECTINS

Lectins seem ubiquitous in nature. They occur in the simplest life forms (viruses) to the most complex (mammalian tissues). In plants, more than 1000 species have been reported to possess lectins. In fact, most plants examined yield lectins or lectinlike activities. There are no rapid-screening methods for all lectins. Because most lectins tend to be multivalent, they generally have the ability to aggregate cells, such as erythrocytes. In exam-ining biological specimens or their extracts for lectins, it must be considered that frequently lectins exhibit a narrow specificity. One kind of red cell may be agglutinated by a lectin, or only the red cells of one or a few species may be susceptible to aggregation. This is because different red cells have unique glycoconjugate compositions and unique distributions of lectin re-ceptors. Nevertheless, hemagglutination is the most reliable and direct

Table 1 Selected Major Experiments in Lectin Research and Lectin-Micro-organism Interactions

Year

1888

1936

1948-52

1957

1960

1960s

1960s

1968-70

1971

1972-73

1973

1973

1977

1978

1979

1984

4

Contributor(s)

Stillmark (1)

Sumner and Howell (14)

Renkonen (15); Bird (16-18)

Makela (19) (also work of Morgan and Watkins; Boyd, Reguera, and oth-ers; further reviewed by Levene et al. (Chapter 10 of this book), Bird (20) and Crookston (21)

Nowell (22)

Goldstein et al. (23)

Kohler et al. (24-27); Wagner (28)

Doyle et al. (29); Goldstein and Staub (30)

Tkacz et al. (31)

Archibald and Coapes (32); Birdsell and Doyle (33)

Doyle et al. (34)

Martinez-Palomo et al. (35)

Ebisu et al. (36)

Stoddart et al. (37)

Schaefer et al. (38)

Graham et al (39)

Observations

Plant extracts could specifically aggluti-nate erythrocytes of various animals.

ConA aggregated members of the gen-era Mycobacterium and Actinomy-ces. Lipid extracts of M. paratubercu-losis were aggregated by ConA.

Developed use of lectins as blood group reagents.

Further studies on blood group antigen interactions with lectins.

Discovery of lectin-induced mitogenesis of lymphocytes.

Specificity of ConA for nonreducing sugar termini shown.

Demonstrated lectin specificity for mi-croorganisms.

ConA was shown to specifically bind li-popolysaccharides of certain gram-negative bacteria.

Identification of budding sites in Sac-charomyces.

ConA blocked binding of bacteriophage to Bacillus subtilis.

First affinity purification of a teichoic acid employing ConA-agarose col-umns.

Lectins employed as probes for patho-genic protozoa.

Lectins were used as structural probes for a streptococcal group-specific polysaccharide.

Identification of fungi in paraffin sec-tions of tissues.

First use of lectins in diagnostic microbi-ology.

Enzyme-linked lectinosorbent assay (ELLA) developed for bacteria and bacterial spores.

Lectin-Microorganism Complexes

Table 1 (Continued)

Year

1984

1985

1988

1989

Contributor(s)

Mobley et al. (40)

Schalla et al. ( 41)

Karayannopoulou et al. (42)

Slifkin and Cumbie (43)

5

Observations

ConA was used to monitor the insertion of and subsequent fate of teichoic acids of Bacillus subtilis.

Lectins were first employed as reagents in the epidemiology of bacterial infec-tious agents.

In situ identification of fungi in tissue sections.

Use of lectins in diagnostic virology with infected tissue cultures.

means of screening for lectins. Furthermore, once a lectin has been shown to clump a particular cell (red cells, fungi, bacteria, or other), the specificity of the lectin can be determined by hapten-inhibition experiments. Knowl-edge of the specificity then frequently leads to affinity purification methods for the isolation of the glycoconjugate-binding proteins. One reason mono-valent lectins have not been discovered may be that there are no rapid means for their detection. It may be possible for monovalent lectins to compete with polyvalent lectins and, thereby, render the latter incapable of causing cellular aggregation, but as far as is known, no systematic search for monovalent lectins has been undertaken. Monovalent lectins would also be expected to be retarded, but not retained, by aff~nity columns.

Table 2 outlines the major sources of lectins. In plants, lectins have been found in the roots, sap, fruit, seeds, flowers, barks, stem, and leaves. Some plants have more than one lectin, and some lectins are synthesized as allelic variants or as isolectins. Some lectins are glycoproteins, but in many instances, the carbohydrate is not required for lectin activity. In bacteria, lectins may occur on the cell surface or may be found in the periplasm (transport proteins) or cytoplasm. The bacterium Pseudomonas aerugi-nosa is the only known prokaryote to express internal lectins [44]. The spectrum of lectin sources is impressive.

For some years, it was a theme of some lectin researchers to find a universal function for the proteins. Now, it seems that the function is related more to the origin of the lectin (Table 3). For example, surface lectins of bacteria are thought to be important in adhesive events. Mutants lacking surface lectins tend to be avirulent [45]. Furthermore, inhibitors of bacteriallectins have been reported to reduce the incidence of experimental infections [46]. Similarly, virallectins (the spikes of influenza viruses are

6

Table 2 Sources of Lectins in Nature

Avian Eggs, serums, tissues

Bacteria

Invertebrates Crustaceans, insects, slugs, snails

Mammals

Doyle

Cell wall Cytoplasm Cytoplasmic membrane Fimbriae (pili)

Eggs, lymphocytes, serum, sperm, various tissues

Outer membrane Peri plasm

Fish, eels, snakes Serums Venoms

Fungi, yeasts, protozoa Surface structures

Plants Flowers Fruit Leaves Roots Saps Seeds Stems

Viruses Bacteriophages Spikes of some animal viruses

the best example) and fungallectins may have roles in adhesion to glycocon-jugates [47]. The influenza virus binds to receptors containing terminal sialic acids. Sialidase treatment of receptor-containing cells renders the cells resistant to the influenza virus. In bacteria, many bacteriophage particles require a-glycosylated teichoic acids as receptors [48].

Etzler [49] has reviewed many of the proposed functional roles for lectins in plants. In one interesting experiment, Marsh [50] grew Dolichos

Table 3 Some Proposed Functional Roles for Lectins

Source(s)

Bacteria, viruses Fungi, molds Nematodes, protozoa Eel and fish serum,

crustacean· tissues Mammalian tissues

Plants

Insects Eggs

Function(s)

Adhesion to glycoconjugates Adhesion; mating factors; differentiation Adhesion; trapping of potential nutrients Primitive antibodies; agglutinins for bacteria

Lectinophagocytosis; removal of desialyated gly-coproteins

Anti-insect; anti-fungal; primitive immune pro-teins; symbiosis; storage protein

Immune factors against protozoa Recognition of sperm glycoconjugates

lectin-Microorganism Complexes 7

biflorus in the presence and absence of blood group A antigen and then analyzed the seeds for the anti-A lectin. Both groups of seeds gave rise to the anti-A lectin, suggesting that the lectin was not synthesized by virtue of antigenic stimulation. It seems likely that lectins of plants are not analogous to antibodies in animals. Plants, however, may not have a need to respond to potential antigens. The lectin may play a more direct role against imme-diate challenges such as from fungi or viruses. Wheat germ agglutinin can inhibit the growth of the plant pathogen Trichoderma viride [51]. Further-more, the lectin can bind to hyphal tips and septa of the fungus. Antifungal properties of the lectin of Solanum tuberosum have been reported. The lectin, which binds oligomers of N-acetylglucosamine, inhibited hyphal ex-tension of Botrytis cinera [52] and caused the release of cytoplasmic constit-uents of Phytophthora injestans [53]. Other studies have appeared that describe antifungal properties of lectins. Lectin from barley is known to reduce the infectivity of barley stripe mosaic virus [54]. In work in the author's laboratory, severallectins capable of binding to bacteria were inca-pable of inhibiting cell division, so it appears unlikely that lectins possess general antibacterial properties. Specific lectins, however, may inhibit se-lected bacteria. If, indeed, lectins do play a general role in reducing the infectiveness of plant pathogens, it must be through an as yet to be deter-mined mechanism.

Lectins of some plants may be toxic to insect predators. The larvae of bruchid beetles are killed by the lectin of Phaseolus vulgaris [55]. For some years, there has been a controversy surrounding the role of lectins in symbi-osis between nitrogen-fixing bacteria and legumes. It is clear that the root lectins of some sprouts of plants can specifically bind nitrogen-fixing bacte-ria. But it is also clear that these same lectins can bind other kinds of microorganisms as well. Furthermore, occasionally, the absence of lectin in mutants of the plants has not led to loss of the ability of the bacteria to adhere to root tips [reviewed in 49].

In animal tissues, some macrophages possess cell surface lectins capa-ble of recognizing bacterial (and possibly other microbial) glycoconjugates. These lectins may be required to achieve nonopsonic phagocytosis. This process has been termed lectinophagocytosis by Ofek and Sharon [56]. Liver cells possess lectins capable of binding asialoglycoproteins. These lectins presumably function in the removal of the glycoproteins from circu-lation. The hepatocyte lectins are sometimes called C-lectins as they are Ca2+ -dependent. The C-lectins bind galactose residues in mammals and are involved in endocytosis of asialoglycoproteins. Another class of animal lectins is called S-lectins. These lectins are soluble in the absence of deter-gents and are usually specific for fl-galactosides. Neither C- nor S-type lectins have been employed in microbiology, as far as is known. Animal

8 Doyle

celllectins [reviewed in 57] are also considered to be involved in egg-sperm recognition, cellular differentiation, metastasis, lymphocyte migration, and hormonal function [a summary of these proposed functions is given in 9]. Lectins have now been firmly established in numerous biological processes. The increasing availability of lectins from many sources provides more probes for the study of microbial and viral glycoconjugates.

Ill. SPECIFICITIES OF LECTINS Traditionally, the specificities of lectins have been defined based on the simplest monosaccharides to inhibit hemagglutination or to bind directly to the protein. Some lectins, however, cannot be inhibited by monosaccha-rides (or disaccharides). For example, the a-1,6-glucan-binding lectin of Streptococcus cricetus cannot be inhibited by high concentrations of iso-maltotriose, but can be inhibited by relatively low concentrations of isomal-tooctaose or higher oligomers [58]. Moreover, peptides contribute to the specificity of some lectins capable of complexing with complex saccharides. Figure 1 shows the structures of most of the sugars and monosaccharides that have been reported to bind with lectins. The figure shows the Haworth structures and, for some, the stable chair conformations. Symbols are in-cluded for the monosaccharides commonly found in polysaccharides or glycoconjugates of bacteria, plants, and animals (all possible monosaccha-rides with symbols cannot be described in this brief overview of lectin specificities, but the figure contains the best-represented ones in the litera-ture). Microorganisms, including viruses for purposes of this book, are known to contain glycoconjugates possessing all the structures shown in Figure 1. In addition, lectins have been reported that interact with microbial and viral glycoconjugates containing the structures shown in the figure. Figure 2 shows ways of presenting some of the oligosaccharide structures known to interact with lectins. Some of the structures are animal cell-derived, but many of the structures are found on viral coats or on cells transformed by viruses (see Chapter 2 for the complete description of viral glycoconjugates capable of interacting with lectins).

Concanavalin A (ConA), the first lectin for which the specificity was studied in detail, binds to unsubstituted nonreducing a-n-glucose (Glc) or a-o-mannose (Man) residues [23]. The basic requirement is that hydroxyls at C-3, C-4, and C-6 must be available. Microbial polymers, such as dex-trans (a-1,6-glucan), which have only one nonreducing o-glucose per chain, bind to ConA, but the protein cannot precipitate with them. Introduction of branches into the linear dextran may result in increased availability of nonreducing termini, leading to precipitation with the lectin. Linear tei-choic acids may precipitate with ConA because of the substitution (in ef-

D·GLUCOSE

D-GALACTOSE

D·MANNOSE

L-FUCOSE

N-ACETYL-0-GLUCOSAMINE

N-ACETYL-0-GALACTOSAMINE

N-ACETYLNEURAMINIC ACID (SIALIC ACID)

Q-NHz

0-GLUCOSAMINE ~OH

4ft~ N-£-CH,

0 N·ACETYLMURAMIC ACID

l\ml m)_(

OH

b.---OH

~~\ HO~

OH

Q--

0

•

-• D

•

METHYL-a-D-GLUCOSIDE OH

METHJ_!.-JI·D-GLUCOSIDE

HO.k\,011 ~

OH OH

a·L·RHAMNOSE

Q~ OH

D·GALACTURONIC ACID

Figure 1 Lectin-reactive carbohydrate structures. The figure shows the most com-mon structure known to complex with lectins. Symbols for some of the structures are shown to the very right. The figure also shows Haworth (pyranose) and chair conformations for many of the carbohydrates. (For carbohydrate structures fre-quently found on glycoproteins; see Fig. 2. Modified from Ref. 9.)

9

10

Mana 3 (Mana6) Man

Mana 6 '-Man Mana 3/

ManC< 2 Mana 3 [Mana 3 (Mana 6) ManC< 6]Man~4GicNAci}4GicNAc

ManC< 6-.........._ /ManaS-........._

Mana 3 __,., Man~4GicNAcj}4GicNAc Mana2Mana3.......-

GaiJl4GicNAcJl2Mana3(GaiJl4GicNAcJl2Mana6)ManJl4GicNAcJl4(L-Fuca6)GicNAc

Doyle

L-FUCC<6 GaiJl4GicNAc~2ManC<6 -- I

o-e-•, .,. o-•-•/•-•-• -- Man~4GicNAcJl4GicNAc

Ga1Jl4GicNAc~2Mana3

GlcNAcJl2Mana3[GicNAc~2(GicNAcJl6)Mana6]ManJl4GicNAc~GicNAc

GlcNAc~6 --.... GlcNAc~2ManC<6 > Man~4GicNAcJl4GicNAc GlcNAcJl2Mana3

GlcNAc~2Mana3(GicNAcJl4)(GicNAc~2Mana6)Man~4GicNAc~4GicNAc ·-· '\ GlcNAc~2Mana6 ·-·-·-· GlcNAc~ -- Man~4GicNAc~4GicNAc / GlcNAc~2Mana3 :__..;;;;-"' ·-·

GlcNAcJl2Mana3[Mana3(Mana6)Mana6]Man~4GicNAc~4GicNAc

Mana6 -- Mana6 ManC<3--------- ... -~----. ManJl4GicNAcJl4GicNAc GlcNAcJl2Mana3---

Figure 2 Representation of lectin-reactive sites commonly found on glycoproteins. The saccharides may be derived from animal cells, viral-transformed cells, or micro-organisms, such as fungi. Lectins frequently complex with oligosaccharides in glyco-proteins. Usually, the most important residues are the nonreducing termini and their accompanying penultimate residues. (Modified from Ref. 9.)

feet, a type of branching) of a-D-glucose residues on the glycerol or ribitol moiety (a later section describes the microbial structures that may interact with lectins). Mannans and glycoproteins may contain a-1,2-mannose link-ages that readily interact with ConA.

The Appendix lists the most common lectins studied to date. Specifici-ties are given in terms of monosaccharides or oligosaccharides that best complex with the lectins. For many of the lectins, only monosaccharides have been studied as inhibitors, whereas for others, inhibitors have yet to


be discovered. The table also lists the common name of the lectin source and, when known, the blood group specificity. A new means for the abbre-viation of lectins is proposed as well. Some common abbreviations, such as WGA, for wheat germ agglutinin, are too well entrenched in the literature to propose a change. When possible, abbreviations should begin with the first two letters of the genus, followed by the first letter of species. When abbreviations overlap, multiple letters of both the genus and species should be employed. Throughout this book, the abbreviations shown in the Ap-pendix will be employed.

An inspection of the specificities of the lectins listed in the Appendix reveals that numerous lectins bind Gal or GalNAc residues. A few of the lectins are specific for anomeric linkages, although most Gal or GalNAc-binding lectins can complex with either a- or ~-linked saccharides. Impor-tantly, although a particular lectin may bind a particular saccharide, there is no certainty that the lectin will bind those residues on a microbial surface. Frequently, hydrophobic residues enhance the interaction between a sac-charide and a lectin. Probably the most unimportant hydroxyl group recog-nized by lectins is C-2. For example, ConA can bind mannose or glucose, monosaccharides that differ only in the spatial orientation of the C-2 hy-droxyl. In general, galactose-specific lectins have no affinity for glucose, and vice versa, showing the role of C-4 in lectin recognition. For many lectins, the penultimate saccharide has a large influence on binding. For example, the lectin from Eranthis hyemalis (ERH) can bind Gal~-1,4GlcNAc somewhat better than Gal~-1,4Glc. Similarly, the Japanese pa-goda tree lectin (SOJ, from Sophora japonica) binds Gal~-1, 3GalNAc somewhat better than Gal~-1,3GlcNAc. Most GlcNAc-binding lectins com-bine best with oligomers of GlcNAc, although as far as is known, all of these lectins can complex with GlcNAc alone. In the Appendix, it should be noted that there is a paucity of ~-glucose-binding lectins. A lectin from the fungus Sclerotium roljsii (SCR) has been reported to bind Glc~-1,3Glc. In addition, Cytisus sessifolius (CSS) has a weak affinity for cellobiose (Glc~-1,4Glc). Lectins capable of binding ~-glucosides would be welcome in diagnostic microbiology, as many bacteria and fungi produce ~-linked

glucosidic polymers. The so-called ~-lectins [69] are known to interact with hydrophobic ~-glucosides, but these have not yet proved to be of value in microbiology. There are also only a few lectins specific for uronic acids. The Ap/ysia depilans (APD) lectin is inhibited by galacturonic acid, but this lectin also binds galactose. The lectin from Abramis brama (ABD) has been reported to complex with rhamnose (Rha) residues, but this lectin also binds galactose. The very few lectins reported to be specific for ~-o-glucose, uronic acids, or rhamnose may reflect that researchers have not looked for such specificities. The ubiquitous occurrence of ~-o-glucose, a-L-rhamnose, and uronic acids suggests that lectins are available that can bind these

12 Doyle

monosaccharides. Lectins specific for sialic acids frequently complex with N-acetylneuraminic acid (NeuAc), whereas others may complex with 9-0-acetylneuraminic acid or N-glycolylneuraminic acid. Only a few bacteria make sialic acid-containing polymers, but many viral protein coats contain sialoglycoproteins that can interact with lectins. Some lectins interact pri-marily with mucin-type or 0-linked glycoconjugates, including the lectins from Agaricus bisporus and Bauhinia purpurea. Other lectins seem to have high affinities for N-linked glycoconjugates, such as the lectin from Ricinus communis. The now widespread availability of numerous lectins increases the probability that lectins will be applied more extensively to the study of microbial surfaces.

IV. THE HYDROPHOBIC EFFECT AND LECTINS

Severallectins are known to possess multiple-combining sites. In addition to saccharide-specific sites, lectins may bind metal ions and hydropho-bic ligands. A common occurrence in legume lectins is a hydrophobic-combining site spatially separated from the saccharide-combining site. In addition, there are hydrophobic sites very near saccharide-specific sites. Concanavalin A can bind p-nitrophenyl-a-o-mannoside better than it can bind methyl-a-o-mannoside which, in turn, is complexed better than a-D-mannose. Also, ConA has a site specific for hydrophobic groups, such as inositol, -adenine, and various fluorescent dyes. Frequently, the affinity constant for the binding of a hydrophobic group is greater than the affinity constant between the lectin and its specific monosaccharide. Microorgan-isms have numerous hydrophobins [70] on their surfaces, including pro-teins, glycolipids, lipoteichoic acids, and others. These hydrophobins, which may or may not be associated with glycoconjugates, no doubt con-tribute to the ability of a lectin to bind to a microbial surface. It is interest-ing that, among the legumes, the hydrophobic cleft has been largely con-served throughout evolution, suggesting a functional role for the site. Hydrophobic sites adjacent to carbohydrate-specific sites also extend to unrelated species. Pseudomonas aeruginosa lectins bind hydrophobic sac-charides better than unsubstituted saccharides. Similarly, the mannose-specific lectin of Escherichia coli has a much higher affinity for hydropho-bic mannosides than for hydroxylated mannosides [71].

V. ISOLATION AND PURIFICATION OF LECTINS

There are no general strategies for the isolation of lectins. The isolation procedure(s) are usually dictated by the source (seed, serum, bacteria, or other) and may involve classic protein purification schema. Some extracts


may be initially fractionated by ammonium sulfate and then by ion-exchange chromatography. If the specificity of a lectin is known, the lectin may be purified to homogeneity on affinity sorbents. Elution of the lectin can then be realized by use of solutions of saccharides or chaotropes, or by lowering the pH. It is essential that eluting agents be removed from the purified lectin(s) and that the agents do not irreversibly alter the saccharide-binding site(s). Affinity methods rarely separate isolectins, although a com-bination of affinity and ion-exchange techniques may afford reasonable separations. For studies on microbial surfaces, it is best that pure lectins be employed when possible. This is because many sources of lectins yield two or more carbohydrate-binding proteins with distinct specificities. The book by Liener et al. [8] provides extensive discussions on the methods involved in lectin isolation and purifications.

VI. LECTIN DERIVATIVES IN MICROBIOLOGY

The microbiological applications of lectins frequently require that the lectin possess a sensitive tag or reporter. Lectins, as proteins, can be derivatized with any of the same reagents that have been employed for antibodies or enzymes [72]. Fluorescein isothiocyanate (FITC) derivatives of lectins have been used in microbiology to detect microorganisms and spores [73,74] and to study the distribution of wall polymers [75]. Figure 3 shows an FITC derivative of soybean agglutinin binding to spores of Bacillus anthracis, a gram-positive organism possessing a cell wall polysaccharide with terminal D-galactose residues. The obvious goal of introducing a marker onto the lectin is to be able to monitor glycoconjugates on microbial surfaces or in solution. It must be remembered that the chemical modification of a lectin may led to a partial loss of its activity. Consequently, chemical modifica-tions are normally performed in the presence of specific saccharides in an effort to prevent inactivation of the combining sites.

Many lectins bind directly onto latex particles, resulting in passively sensitized spheres that can be used in aggregation reactions (Table 4). La-tex-sensitized particles also have been used in establishing specificities of lectins. Such sensitized particles may be susceptible to aggregation by mi-crobial glycoconjugates. Lectins may be coupled with enzymes, thereby permitting enzyme-linked lectinsorbent assays (ELLA). The ELLA tech-niques have been used successfully to detect low densities of bacteria and bacterial spores [39]. Salt-enhanced ELLA assays (SELLA) take advantage of the fact that many substrata for lectins can be salted-out onto plastics. The SELLA techniques make it possible to detect very low concentrations of microbial glycoconjugates. The acronym PELLA is reserved for the use of fluorescent derivatives of lectins, whereas GELLA is proposed to refer

14 Doyle

Figure 3 Binding of fluorescein-labeled soybean agglutinin (SBA) with Bacillus anthracis spores. Vegetative cells also bind SBA. The SBA-reactive material on the spore surfaces may or may not be similar in structure to that on vegetative cell walls. (From Ref. 76.)

to lectin-gold mixtures as probes for glycoconjugates. A main advantage for using dot-blot-like assays with lectin-colloidal gold is that glycoconju-gates can be detected on membrane filters and very low concentrations of gold can be detected visually. The lectin-colloidal gold (GELLA) assays are convenient for the screening of large numbers of samples for glycocon-jugates.

VII. MICROBIAL SUBSTRATA FOR LECTINS

There are numerous microbial structures that serve as receptors for lectins. Most of the lectin-reactive glycoconjugates are listed in Table 5. In bacteria, cell wall- or outer membrane-associated components are the most common lectin receptors. Teichoic acids, covalently linked to peptidoglycans, occur in many gram-positive bacteria. Polymers of glycerol phosphate or ribitol phosphate may contain a- or 13-linked carbohydrate substitutions on the carbons not attached to phosphates. In B. subtilis W23, a /3-D-glucosyl unit is attached to the C-2, 3, or 4 groups of the ribitol, whereas for B. subtilis


Table 4 Lectins, Lectin Derivatives, and Procedures Involving Lectins in Micro-biology

Methods

Agglutination (direct) Agglutination (indirect)

BELLA

Enzyme-linked lectinsor-bent assay (ELLA)

FELLA

GEL LA

RELLA

SELLA

WELL A

Enzyme assays

Lectin in combination with flu-orogenic or chromogenic substrate

Lectinophoresis

Description

Soluble lectins bind to microbial surface. Latex spheres are passively sensitized with lee-

tins. Biotin-conjugated lectin may be detected by an

avidin-enzyme conjugate. Lectin may bind to plastic, or lectin may be used

to complex with plastic-bound microbe or an-tigen.

Fluorescent lectin is used to detect glycoconju-gates or microbes.

Lectin-colloidal gold mixture detects low densi-ties of microbes or low concentrations of glyco-conjugates.

Lectin may be derivatized to contain radioactive 3H, 14C, 1311, or 1251.

Salt-enhanced enzyme-linked lectin-sorbent assay; ammonium sulfate promotes binding of lectin or protein antigen to polysty-rene.

Western blot modified so a lectin can bind to a macromolecule in a gel.

Lectin is coupled to enzyme, such as ,8-galactosidase or a peroxidase.

Lectin may detect one kind of bacterium, but con-ventional fluorogenic or chromogenic sub-strate may detect closely related bacteria.

Lectin is substituted for antibody in rocket elec-trophoresis.

168, the C-2 of the glycerol contains a-o-glucosyl substitutions. As indi-cated in the foregoing, ConA and other a-o-Gle-specific lectins can com-bine with the teichoic acid. Figure 4 shows an example of a teichoic acid structure, along with some other microbial polymers known to be reactive with lectins. Some strains of Staphylococcus aureus possess ribitol phos-phate teichoic acids that are substituted with both a- and {3-GlcNAc. These teichoic acids interact with ConA and WGA, respectively.

Muramic acid (usually in the N-acetylated form) will react weakly with some GlcNAc- and sialic acid-binding proteins. Most muramic acid residues possess amino acid substitutions on the lactyl groups on C-3, so in pepti-

16

Table 5 Lectin-Reactive Sites on Microorganisms and Viruses

Bacteria Capsules Glycolipids Glycoproteins (infrequent) Group-specific polysaccharides Levans (polyfructans) Lipomannans Lipooligosaccharides Lipopolysaccharides Lipoteichoic acids Peptidoglycans Surface array layers Teichoic acids Teichuronic acids Type-specific polysaccharides•

Fungi Arabinans Capsules Chitin Galactans Glucans Glycoproteins Mannans

Protozoa Galactomannans Glycolipids Glycoproteins Lipophosphoglycans Phosphoglycans

Viruses Envelope glycoproteins

•Types of polysaccharides that occur in oral streptococci are not to be confused with type-specific M-protein of pyogenic cocci.

Doyle

doglycans, muramic acids generally form poor receptors. Peptidoglycans, however, are able to complex with WGA, presumably through interaction with nonreducing GlcNAc termini [77].

The lipopolysaccharide shown in Figure 4 would be expected to inter-act with WGA and galactose-binding lectins. Connelly and Allen [78] and Allen et al. [79] showed that a battery of lectins could be used in structural determinations of lipolysaccharides from Neisseria gonorrhoeae. Doyle et al. [29] found that Shigella f/exneri lipopolysaccharides could precipitate with ConA. Several other lipopolysaccharides formed weak complexes with the lectin. Goldstein and Staub [30] observed that although some lipopoly-saccharides possessed the requisite terminal a-n-glucose residues, they would not precipitate with ConA. These findings suggested that penulti-mate or nearest-neighbor residues may influence the interaction with the lectin. Mutants in lipopolysaccharide oligosaccharide structures may result in exposed or lost lectin reactive sites. Hammarstrom et al. [80] have studied the reactivity of lipopolysaccharides from a Salmonella sp. and found that the extent of mutation may lead to various lectin reactivities. For example, the wild-type salmonellar lipopolysaccharide was unreactive with Helix po-matia agglutinin (HPA; see Appendix), but mutations leading to "rough" colonies gave rise to a product capable of interaction with the lectin, and a final "deep rough" mutant was again unreactive. The lipopolysaccharides


0 II H H H

- 0 - p - 0 - c -c -c - 0 -

A teichoic acid I H I H 0- n a-D-Gic

EthN-P

I KDO

A lipopolysaccharide I

I Lipid AI- Glc-Gai-Gic Hep-Hep-KDO-KDO ( Man-Rhm-Gal )0

I I I GlcNAc Gal P-P-EthN

A dextran (a-1 ,6 Glc)n

A peptidoglycan -(MurNAcP-1, 4 GlcNAcp)-I n peptide(s)

Chitin ( GlcNAc P - 1, 4 ln

A capsular polysaccharide (GicU p- 1 ,4 Glc)n

Figure 4 Potential lectin receptors derived from microorganisms. Teichoic acids are covalently bound to peptidoglycan of many gram-positive bacteria. Lipoteichoic acids (L T A) are anchored in cell membranes and not associated with the walls of most (group A streptococci excepted) gram-positive cells. The L TAs, consisting of a chain of poly(glycerol phosphate) may be glycosylated, similar to wall teichoic acids. Lipopolysaccharide (LPS) structures are dependent on the genus or species of the organism producing them. Capsular polysaccharides also exhibit diversities of structures, either from gram-positive or gram-negative microorganisms. The sim-plified structures shown in the figure do not represent all potential lectin-reactive materials produced by microorganisms. A more complete list is given in Table 5.

18 Doyle

of many gram-negative bacteria possess limulin-reactive 2-keto-3-deoxy-octonate (KDO; see Fig. 4) [81]. Gilbride and Pistole [82] have suggested that limulin may serve as a type of immune factor in the horseshoe crab, because of the ability of the lectin to bind lipopolysaccharides.

Branched dextrans (a-glucans) precipitate with ConA and other a-glucose-binding lectins. Increased branching, leading to a greater propor-tion of nonreducing glucose termini, enhances reactivity with ConA [23]. Therefore, ConA is a tool for studying a-glucan structures. Mannans, lev-ans, galactans, arabinogalactans, galactomannans, various group-specific polysaccharides of streptococci, and teichuronic acids have been reported to bind with one or more lectins. An acidic lipomannan from Micrococcus luteus has been detected by ConA in lectinophoresis [83] (see Table 4), a type of rocket electrophoresis substituting lectin for antibody.

Several reports describe the interaction between bacterial teichoic acids and lectins [84-86]. Precipitin reactions in gels have been employed to detect complex formation between ConA and teichoic acids from members of the genus Bacillus [87]. In addition, soluble teichoic acids are precipi-tated by ConA. Cell wall-bound teichoic acids can be detected on the sur-faces of the bacilli by FITC-ConA. Lectins from Triticum vulgaris (WGA) and Helix pomatia (HP A) have been used to study cell wall teichoic acids from staphylococci. Reeder and Ekstedt [85] found that an a-o-gluco-sylated teichoic acid from a strain of Staph. epidermidis was precipitated by ConA. This was unusual because most teichoic acids from Staph. epider-midis strains are not a-o-glucosylated. Also, ConA can be used as a probe for the surface teichoic acid of Streptococcus jaecalis (Enterococcus hirae) [87]. When the teichoic acid is removed by extraction with acids, the reac-tivity of Strep. jaecalis with ConA is abolished.

Individual chapters in this book will provide details on interactions between lectins and various specific surface glycoconjugates of microorgan-isms and fungi. The reactivity of a lectin with a particular glycoconjugate depends on several factors (Table 6). For example, lectin molecular weights vary from a few thousand to over one-half million. Therefore, some lectins can penetrate to sites inaccessible to antibodies or other higher molecular weight lectins. Furthermore, some glycoconjugates may be masked, render-ing them inaccessible to any lectin. An example is the peptidoglycan of certain bacteria that is not available to lectins because of outer membranes or capsules. Dextran-covered streptococci could be made ConA-reactive by incubation of the cells with dextranase [88; see also 89]. In Mycoplasma, lectin-reactive sites are exposed by treating the bacteria with proteases [90]. In addition, salts may decrease the interaction between lectins and glyco-conjugates by causing an unfavorable conformation in the glycoconjugate [91]. Work in my laboratory has shown that some bacteria become lectin-

Lectin-Microorganism Complexes

Table 6 Some Factors Governing the Reactivity of Microbial Glycoconju-gates with Lectins

Factors

Lectin molecular weight (MW)

Adjacent hydrophobic resi-dues

Presence of salts

Cell wall turnover

Time of interaction

Location of lectin- reactive site(s)

Proteases

Comment

High MW lectins may be excluded from carbohydrate receptors.

Some lectins preferentially bind to sites near hydrophobic groups.

Salts induce a rigid-rod to random coil conformation in teichoic acids, thereby diminishing the rate of bind-ing with lectins.

In some bacteria, lectin-reactive sites are shed into medium ("turned over").

In microorganisms with a low density of lectin receptor, .considerable time may be required to detect an interac-tion.

Some glycoconjugates may be masked by capsule, outer membrane, peptid-oglycan, or other factor, thereby pre-venting their accessibility to lectins.

Proteases may expose (or abolish) lec-tin-reactive sites on microorganisms.

19

reactive only after prolonged incubation times with the protein. Hydropho-bic interactions may play a role in complex formation between lectins and glycoconjugates. For example, a-glucosylated lipoteichoic acids of B. subti-lis cannot be readily eluted from ConA-Sepharose columns unless mild chaotropes are present along with methyl-a-o-mannoside. Finally, some microorganisms shed their cell surface components during growth. These shed or turned over materials may not be replaced, resulting in loss of lectin-reactive sites [92].

VIII. APPLICATIONS OF LECTINS IN MICROBIOLOGY

Lectins have now become essential tools for the study of microbial glyco-conjugates. A few of the uses of lectins in microbiology were given in Table 1, where some of the historical aspects of lectin-microorganism interactions were listed. The purpose of this section is to provide an overview of the applications of lectins in various practical and research problems related to

20 Doyle

microbiology. Table 7 lists some of the most common applications of lee-tins in microbiology reported to date.

In diagnostic microbiology, lectins have now become standard re-agents. In a typical application, a suspension of a microorganism may be mixed with a solution of lectin on a glass slide. An agglutination can often be taken as a confirmation of an organism, providing that some knowledge of the history of the organism is available. For example, N. gonorrhoeae can be confirmed by agglutinating with WGA, assuming the isolate came from a urethral exudate and was a gram-negative diplococcus. If the isolate originated from a spinal tap, a throat swab, or the skin, WGA may be expected to aggregate a few strains of N. menigitidis or N. lactamica. The agglutination of N. gonorrhoeae by WGA requires a microgram or less of the lectin, whereas the other bacteria are usually agglutinated by higher concentrations. In fact, one of the advantages of employing lectins as diag-nostic reagents is that they are generally active at very low concentrations. Moreover, agglutination reactions are usually rapid. Th~ use of lectin deriv-atives (see Table 4) potentiates the possibilities for lectin applications in diagnostic procedures. The detection of herpesviruses in tissue cultures [43] and the detection of infectious agents in tissue sections [42] are good exam-ples of how derivatized lectins can be used in diagnostic microbiology pro-cedures. Table 8 offers an outline of some of the major uses of lectins in diagnostic procedures. Slifkin, Chapter 4 in this book, provides a compre-hensive review of the uses of lectins in clinical diagnostic microbiology.

Table 7 General Applications of Lectins in Microbiology

Affinity sorbents for microbial polymers and microbial products Detection of intracellular viruses and microorganisms Detection of microorganisms in situ Detection of mutants in surface glycoconjugates Probes for monitoring insertion and fate of cell surface glycoconjugates Probes for solution properties of polyelectrolytes Purification of immunoglobulins Reagents for diagnostic microbiology Reagents to be employed in establishing structures of glycoconjugates Reagents to establish epidemiological patterns of infectious agents Study of symbiosis between bacteria and sponges Reagents that can be employed to determine receptor identities for bacteriophages Selective inhibitors of enzymes Studies on adhesion mechanisms of microorganisms Use in identification of antigens, including blood group antigens

Source: Details may be found in Refs. 93-111.

Table 8 Summary of Some Specific Applications of Lectins in Diagnostic Microbi-ology and Epidemiology

The pathogen Bacillus anthracis can be distinguished from other bacilli by its growth at 37°C and aggregation with the Glycine max lectin.

Neisseria gonorrhoeae and nonencapsulated N. meningtidis are selectively aggluti-nated by low concentrations of wheat germ agglutinin.

Serogroup A Campylobacter fetus could be correctly identified with lectins. Group B streptococci can be specifically agglutinated by lectins bound to polysty-

rene particles. Cross-reactivity with streptococcal groups A,C,D,F, and G was not observed. A lectin from Cepaea hortensis is specific for group B streptococci.

Group C streptoccal antigen is selectively agglutinated with a lectin from Dolichos biflorus bound to polystyrene particles.

Enzyme-linked lectinsorbent (ELLA) assays can be used to detect low numbers of Bacillus anthracis.

Surface antigen of Streptococcus faecal is isolates from endocarditis patients could be identified by blotting techniques using lectins.

Most common isolates of Listeria monocytogenes could be grouped with a battery of lectins.

Fluorescein-conjugated lectins selectively bound to microbial isolates from the hu-man cornea.

Fungi could be directly observed in tissue sections by use of lectins. Rhabdoviruses of plants could be distinguished from plant tissue by use of three lee-

tins. Subtypes of Marek disease virus could be discriminated by a battery of lectins. Lectins could discriminate between pathogenic and nonpathogenic South American

trypanosomes Smooth and rough strains of Brucella sp. displayed unique agglutination patterns

with lectins. Fluorescein-labeled Helix pomatia lectin could rapidly distinguish herpes simplex

types I and 2 in cell culture. Thermophilic Campylobacter sp. demonstrated unique reactivities with a battery of

lectins and plant agglutinins. A battery of lectins was used to distinguish between Neisseria gonorrhoeae isolates

from various geographic locations. Campylobacter jejuni and C. coli were grouped according to their reactivities with

severallectins. Isolates of Hemophilus ducreyi from different geographic areas gave rise to unique

agglutination patterns with lectins Wheat germ agglutinin has been proposed as a reagent to discriminate between

gram-positive and gram-negative bacteria.

Source: Refs. 11-13, 110-128, 131, 132. Chapter 10 describes the use of individuallectins in blood banking.

22 Doyle

Levy [133] showed that 2-10 ~g/ml of WGA blocked attachment of Chlamydia psittaci to mouse fibroblasts. The blockage could be inhibited by GlcNAc, but not other saccharides or sugars. Moreover, ConA and RCA-1,11 were unable to block the chlamydial attachment. A strain of C. trachoma/is, isolated from a lymphogranuloma venereum lesion, was also prevented from adhering to the fibroblasts by WGA. Identification of car-bohydrates involved in bacterium-animal cell interaction may lead to new means of preventing certain infections. An agglutinin (lectinlike substance of unknown composition) from Persea americana prevents the adhesion of Strep. mutans to saliva-coated hydroxylapatite [98]. The saliva-coated hydroxylapatite (S-HA) serves as a model for tooth surfaces, so consider-able efforts have been made to prevent attachment of cariogenic strepto-cocci to the surfaces of the S-HA beads. Streptococcus downei (formerly Strep. mutans serotype h) adhesion to the S-HA was not inhibited by vari-ous carbohydrates, but the binding was abolished by a protease. The bacte-ria had to be pretreated with P. americana agglutinin to achieve significant inhibition of adhesion. Halverson and Stacey [95] have employed various lectins as mediators of adhesion between Rhizobium (Bradyrhizobium) ja-ponicum and soybean root. A mutant of R. japonicum, incapable of caus-ing rood modulation, could be made phenotypically wild-type (modulation-positive) by very low concentrations of soybean agglutinin (SBA). Concentrations of SBA as low as ten molecules per bacterium were effective in restoring the wild-type characteristics. The results suggested that modula-tion was dependent on adhesion of the bacterium to the root surface. Ef-forts are underway in several laboratories to study the role of lectins in mediating attachment of bacteria to plant tissues. Electron microscopic techniques, employing SBA-ferritin, were used to localize the lectin binding site on Rhizobium japonicum. The SBA-ferritin binds to a capsular poly-saccharide at one end of the cell [134]. The capsular polysaccharide was not contaminated with lipopolysaccharide, nor did outer membrane bind with the lectin. The polysaccharide is a good candidate for bridging between nitrogen-fixing bacteria and legume root tip lectins. Furthermore, the local-ization of capsule at one end of the bacterium raises an important question in bacteria physiology about sites of secretion of exopolymers.

The capsular polysaccharides of pneumococci vary considerably in composition and linkages. The pneumococcal S-14 polysaccharide studied by Lindberg et al. [135] was proposed to have the following structure

- GlcNAc{3-1 ,3Gal{3-1 ,3Glc{3-1 ,6-Gal/3-1 ,3

Ebisu et al. [36] found that WGA and RCA-I, II could precipitate the S-14 polysaccharide, but the a-D-Gal-specific lectin from Griffonia (Bandeiraea) simplicifolia could not. The lack of reactivity with the griffonial lectin


confirmed the existence of the ~-o-galactose terminal linkage. A linear polymer of S-14 could be derived by periodate oxidation, followed by Smith degradation, which removes the ~-o-galactosyl residues. The linear polymer retained its ability to bind with WGA, but its reaction with RCA was lost, as expected. These results show the versatility of lectins in structural work on bacterial glycoconjugates.

Wheat germ agglutinin has an affinity for sialic acid residues, as well as ~-linked GlcNAc residues. Gray et al. [136] took advantage of the reac-tivity of WGA for sialic acids to aid in the purification of streptococcal group B, type-specific polysaccharides. The type-specific, but not the group-specific, polysaccharide, could be eluted from WGA-Sepharose col-umns in reasonably high yield and free of contaminants.

Lectin bound to magnetic microspheres has also been employed in detecting and concentrating bacteria in dilute suspensions. Patchett et al. [109] coated spheres with the H. pomatia lectin and observed that strains of Listeria monocytogenes would bind avidly to the lectin-sphere conju-gate. They further showed that L. monocytogenes would adhere to HPA-agarose columns, only to be eluted by GalNAc. The column and sphere techniques made it possible to concentrate L. monocytogenes from low densities of cells. Such techniques may have value in the food and dairy industries for which L. monocytogenes is a frequent contaminant. Interest-ingly, of several GalNAc-specific lectins, only HPA interacted with most of the strains of L. monocytogenes. Most other food-borne bacteria, such as members of the genera Salmonella, Bacillus, and Staphylococcus, did not complex with HPA.

It has been suggested that lectins can be employed to enumerate yeasts in a suspension [137]. Lectin-conjugates were loaded into plastic syringes, then suspensions of yeasts were poured over the columns. The yeasts were assayed by their ability to produce metabolites, the concentrations of which were proportional to the densities of yeasts bound onto the columns. The method is clever, but there are several items of concern. Some yeasts simply do not interact with any known lectins. Some yeasts, although capable of interacting with a lectin, may not respire or ferment. The method has prom-ise for distinguishing between limited numbers of metabolically active yeasts, but for a pure culture, direct-counting methods would seem supe-rior.

The detection of human immunodeficiency virus (HIV) envelope anti-gens has become important in clinical and hospital laboratories. The anti-gens are generally glycosylated and, therefore, are potentially able to inter-act with lectins. Robinson et al. [138] coated microtiter plates with solutions of ConA, then used the coated wells to trap soluble HIV envelope antigens. The antigens were obtained from detergent-solubilized glycoproteins re-

24 Doyle

leased into the culture medium of HIV-1-infected cells grown in serum-free medium. The HIV antigens, trapped on the solid surface, could then be quantitated by use of enzyme-linked immunosorbent assay (ELISA) tech-niques. No false-positive results occurred among 16 HIV-negative sera, and no false-negative results occurred among 14 HIV-positive sera.

IX. BACTERIAL CELL WALLS, BACTERIOPHAGES, AND LECTINS

Bacterial viruses frequently bind to carbohydrates as a first step in the infective process. In B. subtilis, glucosylated teichoic acids are required for the binding of many bacteriophage particles [48]. The teichoic acid, how-ever, must be covalently bound to peptidoglycan to serve as phage receptor sites. Concanavalin A and bacteriophage t/>25 competed for the same site(s) on the cell wall of B. subtilis 168 [33], a bacterium containing a glucosylated poly(glycerol phosphate) teichoic acid. Table 9 shows that phage t/>25 ab-sorbs to glucosylated walls, but, when ConA is present, no adsorption occurs. Removal of the teichoic acid with 100 mM sodium hydroxide re-sulted in a wall incapable of binding phage t/>25. Soluble glucosylated tei-choic acid did not compete with cell walls, but neutralized the effect of the lectin. Figure 5 shows that ConA, when added to a mixture of walls and phage t/>25, interrupted the adsorption of the virus. When the ConA iiihibi-tor, methyl-a-o-glucopyranoside, was added, adsorption commenced. The ConA may be directly competing with the virus for receptors, or the lectin may cover a composite site consisting of teichoic acid and peptidoglycan. A soluble autolysate or lysozyme digest of the cell wall will not bind to the virus, showing that an intact cell wall containing glucosylated teichoic acid is the actual phage receptor [33]. Concanavalin A not only blocked phage receptor sites on B. subtilis, but also blocked sites on Staph. aureus, provid-ing the staphylococcus possessed nonreducing a-o-N-acetylglucosaminyl res-idues in its teichoic acid [32]. As a control, it was shown that ConA would not alter phage binding to B. subti/is W23, a bacillus possessing ,S-linked o-glucose residues in its teichoic acids.

Lactobacillus casei, another gram-positive rod, possesses a cell surface polysaccharide outside the peptidoglycan that serves as a receptor for bacte-riophage PL-1. L-Rhamnose, a component of the wall-associated polysac-charide, inhibits phage adsorption to the cells [139]. Slight inhibition was observed for o-mannose and L-fucose. A streptomyces hemagglutinin (crude lectin) of anti-B activity (specific for L-rhamnose and o-galactose; see Appendix) inhibits phage binding to cell walls of L. casei [140]. In addition, small reductions in phage binding were noted for o-glucose (or

Table 9 Effects of ConA on Phage Adsorption to Bacillus subtilis Strains

Adsorption (OJo)

Walls Walls +

Hexose/ Walls + teichoic Carbon phosphorus Walls + teichoic acid+

Strain Phenotype source ratio alone ConA acid ConA

168 Trpt/>25S Glucose 0.8:1.0 99.9 0 100 67 Galactose 0.8:1.0 100 0 100 39

gtaB290 Trpt/>25R Glucose 0.06:1.0 0 0 NO NO Galactose 0.04:1.0 0 0 NO NO

gtaC10 Trpt/>25R Glucose 0.8:1.0 0 0 NO NO Galactose 0.30:1.0 99.1 0 NO NO

Strain 168 is wild-type; strain gtaB290 is a mutant unable to glucosylate its wall teichoic acid; strain gtaClO glucosylates its teichoic acid at permissive conditions (growth on galactose). Reaction mixtures contained 100 1-lg cell walls, 2.0 mg ConA, 2.0 mg teichoic acid, 3 x 107 plaque-forming units (pfu) of phage t/125 in a total volume of 1.0 mi. Cell walls were suspended in a minimal medium that contained the concentration of ConA or teichoic acid, or both, desired in 0.9 rnl volume. After 10 min at 30°C, 3.1 x 107 pfu of phage t/125 (0.1 ml) were added, and incubation continued for 15 min. The adsorption was terminated by a 1:100 dilution into cold medium, following by further dilution and plating by the agar-overlay technique. Teichoic acid was from B. subtilis 168. R, resistant; S, sensitive. Source: Ref. 33. ·

[ ~· ~ ;::;· g

~ = ;;· 3 ("l Q 3

"CI

[

N 1.11

26

2

•

4 6 TIME (MIN)

Doyle

• 8 10

Figure 5 Effect of ConA on the kinetics of bacteriophage ¢J25 adsorption to cell walls of B. subtilis 168. The reaction mixtures contained (in a total volume of 1.0 ml) 0.2 mg of cell walls, 2.0 mg of ConA, 1.5 x 107 plaque-forming units of ¢J25, and 0.1 M methyl-a-o-glucopyranoside. At the times indicated, samples were removed, diluted 1 : 100 in cold medium, further diluted, and plated by the agar-overlay procedure. Symbols: 0, untreated cell walls; •, cell walls plus methyl-o:-D-glucopyranoside (a-MG); _., ConA added at zero time; •. ConA added at first arrow and the glucoside added at second arrow. (From Ref. 33.)

D-GlcNAc), but not for lectins that bound only GlcNAc. It was suggested that L-rhamnose was the primary receptor for the phage [140].

X. INTERACTION OF TEICHOIC ACIDS WITH LECTINS

Teichoic acids are frequently substituted with lectin-reactive carbohydrates. In B. subtilis 168, the teichoic acid is a:-n-glucosylated at the glycerol C-2, whereas in B. subtilis W23, 13-n-glucosyl substitutions occur on carbon positions 2, 3, or 4, but these glucose residues are not receptors for readily available lectins. In Staph. aureus, the presence of teichoic acid a:-D-glucosaminyl residues (mostly N-acetylated) renders the cells agglutinable by ConA. Doyle and Birdsell [86] found that double diffusion in agar gels


was a good way to monitor lectin-teichoic acid interactions. They observed that teichoic acids of B. subtilis 168 would form precipitin bands with ConA in agar gels. When the gels were soaked in ConA inhibitors (o-mannose or methyl-a-o-mannopyranoside), the precipitin lines dissolved. Reeder and Ekstedt [85] used a similar method to study ConA-teichoic acid complexes in staphylococci. When soluble B. subtilis 168 teichoic acid was allowed to interact with ConA, typical precipitinlike profiles were ob-tained, characterized by a zone of teichoic acid excess, an equivalence zone, and a zone of ConA excess [91]. Inhibition of precipitation was brought about by the same inhibitors of ConA-neutral polysaccharide complexes. Results suggest that teichoic acids may exist in two conformations. One conformation is random-coil, found in reasonably high salt solutions. The other is rigid-rod, found in dilute salts and buffers. The teichoic acid in the rigid-rod conformation is readily precipitated by ConA, whereas the random-cell teichoic acid is less readily able to interact with the lectin (Fig. 6) [91]. Interaction of teichoic acids with lectins, therefore, is salt-dependent, whereas neutral polysaccharide structure is largely unaffected by salts [91] (Table 10). The rigid-rod conformation of teichoic acids in low-ionic-strength medium is probably due to electrostatic repulsion groups in the teichoic acid backbone structure. Ions would tend to neutral-ize the phosphate groups, resulting in a random-coil conformation. Tei-choic acid conformation may be important in interactions with specific antibodies or autolysin binding to walls. Concanavalin A has proved to be a good probe to distinguish between random-coil and rigid-rod conforma-tions of teichoic acids.

Peptidoglycans of Staph. aureus are receptors of WGA, if the pepti-doglycans possess terminal nonreducing GlcNAc residues [77]. Similarly, staphylococci possessing teichoic acids substituted with {j-GlcNAc residues were good receptors for WGA. As far as is known, there have been no attempts to purify soluble peptidoglycans using WGA affinity sorbents (see also Chapter 8).

Classic preparative schema for cell wall teichoic acids involve extrac-tion of walls with acids or bases. These methods yield polydisperse and impure preparations. Doyle et al. [34] were able to isolate an undegraded (based on physical properties) teichoic acid of B. subtilis 168 by use of ConA-agarose column (Fig. 7). They showed that when autolysates were poured over the ConA column, most of the peptidoglycan and protein emerged near the void volume. The teichoic acid was eluted only by ConA inhibitors. The teichoic acid isolated by ConA affinity chromatography gave a narrow, single band in the analytical ultracentrifuge. In contrast, teichoic acids prepared by conventional extraction procedures were polydis-perse, as revealed by analytical ultracentrifugation. Interestingly, the ConA

28 Doyle

140 2.8 • I T II 120 II 2.4 I

II I II • II

100 II I 2.0

I I E' I I c

80 I 1.6 0 I (\J I (\J I -

E I w ....... 60 I 1.2 0 a. I z I <( Cl I :::L 40 • aJ

I I~ 0.8 a: e I 0 I I C/) I I 20 I I 0.4 aJ

I I <( I I

0 0 20 60 100 140 180 220240

EFFLUENT VOLUME (ml)

Figure 6 Affinity chromatography of a bacterial teichoic acid on ConA-agarose. An autolysate of cell walls of B. subtilis 168 was poured over a ConA-agarose column. The glucosylated teichoic acid was eluted with the addition of methyl-a-D-glucopyranoside to the column (elution with the glucoside began at the arrow). (From Ref. 34.)

column has proved useful in the isolation of mutants deficient in cell wall glucosylation. Teichoic acid, prepared by the affinity method, is a good antigen when mixed with a polymer of opposite charge.

Several other reports document the use of lectin affinity chromatogra-phy for the isolation of teichoic acids. Ndule et al. [141] observed that a small fraction of GlcNAc-containing teichoic acid from Staph. aureus is retained on WGA-Ultrogel. The teichoic acid fraction seemed to partially bind to the column by ionic phenomena. Later, Ndule and Flandrois [142] showed that the wall fraction contained ribitol phosphate, GlcNAc, and alanine. A GlcNAc-containing ribitol-teichoic acid of B. subtilis W23 can be resolved on WGA-Sepharose [143]. Lectin chromatography was useful in the separation of glycerol teichoic acids and mannitol teichoic acids in members of the genus Brevibacterium [144]. The streptococcal group N antigen could be purified as a galactosyllipoteichoic acid [145]. Recently, Leopold and Fischer [146] were able to purify lipoteichoic acids from En-

lectin-Microorganism Complexes

Table 10 Solubilities of Concanavalin A-Teichoic Acid and Conca-navalin A-Glycogen Complexes

Complex

Teichoic acid" Tris (50 mM, pH 7 .5) NaCl (100 mM) NaCl (1.0 M) KCI (1.0 M) Galactose (100 mM) Methyl-a-o-mannopyranoside (100 mM)

Glycogen Tris (50 mM, pH 7.5) NaCl (l.OM) Galactose ( 100 mM) Methyl-a-D-mannopyranoside (100 mM)

Soluble ConA (p.g/4 ml)

49 390 645 580

55 827

33 50 53

761

Complexes were from reaction mixtures containing 1.0 mg ConA, 1.0 mg B. subtilis 168 teichoic acid, or 2.0 mg rabbit liver glycogen, in 50 mM Tris (pH 7 .5) in 2.0 ml volumes. Following incubation for 2 hr at 3 °C, the precipitates were collected by centrigugation, washed twice with Tris, and finally, sus-pended in 4 ml of the indicated solvents. Soluble protein was measured following an additional2 hr incubation at room temperature. •For teichoic acid-ConA complexes, radioactive ConA was employed, the soluble contents of which were determined by scintillation counting. For ConA-glycogen complexes the soluble ConA was assayed by a colorimetric method. Source: Ref. 91.

29

terococcus jaeca/is, E. hirae, and Leuconostoc mesenteroides on columns containing ConA. Interestingly, the LTAs were shown to be heterogeneous in the extent of their glucosylation and chain length. The amounts of ala-nine ester in the L T A were uniform. Furthermore, the ester-linked alanine and the glucose moieties were found on the same poly(glycerol phosphate) chains.

Various papers in the 1960s and early 1970s suggested that cell wall teichoic acids were arranged on the outer surface of the gram-positive cell wall. The appearance of electron-dense outer regions of the walls gave rise to the notion that the teichoic acids were distributed asymmetrically. Doyle et al. [75] found that when walls were partially autolyzed, the walls bound more ConA (Fig. 8). The amount of ConA bound to a partially autolyzed wall was greater than the lectin bound to native walls. However, the relative amount of hexose remaining in the wall was virtually constant, a finding

30

1.0

0.8

"0 -~0.6 0> E

~ 0.4 0 u

0.2

0.125 0.25 0.5 TEICHOIC ACID (mg)

c 0.75

/ I I

1.5

Doyle

3.0

Figure 7 Precipitin profile between ConA and the teichoic acid of B. subtilis 168. ConA (1.0 mg), with the indicated concentration of teichoic acid (2.0 ml final vol-ume), was incubated for 2 hr at 25°C. The precipitates were removed by centrifuga-tion, washed once with 5 ml of the indicated solvent, and analyzed for protein content. (A), 50 mM tris(hydroxylmethylamino)methane (Tris), plus 1 mM Mg2+,

pH 7.5; (B), 50 mM Tris; (C), 50 mM Tris plus 1M sodium chloride. (From Ref. 91.)

that suggested that walls were "loosened" by autolysis, making masked receptors available for interaction with the lectin. Calculations suggested that at least one-half of the teichoic acid of B. subtilis 168 was intercalated within the wall matrix and available for interaction with the lectin only after autolysis (or lysozyme digestion).

Anderson et al. [147], Mobley et al. [40] Kirchner et al. [148], and Kemper et al. [149] applied Fl-ConA to the study of surface expansion in bacterial gram-positive rods, particularly B. subtilis. For several years, it was a mystery how B. subtilis expanded its surface during division. Several authors assumed that surface expansion in bacilli was analogous to that of streptococci, where a single growth zone defined the boundary of expan-sion. Mobley et al. [40] were able to make use of several known obser-vations on the cell walls and teichoic acids of B. subtilis. First, ConA specifically and reversibly binds to a-D-glucosylated teichoic acids. Second, teichoic acids and peptidoglycan are coordinately assembled in B. subtilis.


Finally, phosphoglucomutase mutants (gtfC) cannot glucosylate their tei-choic acids. Mobley et al. used fluorescein-labeled ConA to study the inser-tion and fate of cell wall in temperature-sensitive gtfC mutants of B. subti-lis. They found (Fig. 9) that when ConA-reactive cells, grown at the permissive temperature, were shifted to the nonpermissive temperature, the lectin-reactive sites disappeared randomly over the cell cylinder surfaces, but were retained in the polar areas. In contrast, when cells were shifted from nonpermissive to permissive conditions, ConA-reactive sites (new wall) were found very early in cell septa, but appeared diffusely in cell cylinders (Fig. 10). Old poles did not bind the lectin at all for several generations. The results were taken as evidence !or the diffuse intercalation of wall in the cell side walls during division process. Poles (matured septa) were considered to be assembled in a manner analogous to that for strepto-cocci. Side walls seemed to elongate because of the random (or diffuse) addition of new wall polymers on the face of the wall near the plasma

s:: (/) • 0.9 0 0 .. -·----.---.-------- r r 0.8 ~ c

m 0.24 17 0.7 ~ r m

'1 '--l -u 0.6 6 I 0.20 J: 100 ~

E _,... m -u

c ? 15 "' ~ X I

0 0 / 0 0 0 0.16 / (f) 80 IJ <D / [!1 c w / , (f)

u /. I ~ / 0 z 0.12 ~ 13 /

c{ II/ (f) 60 2. -u ID u / I s:: a: -l..-.; / 0 N 0 Iii (f) 0.08 ~ / ' IJ 3' ID /{ --. c 40

c{ ' ' !!J c 11 / ' 2 / ...... /

f 0.04 / /I ...... -- 4.. 20

)'' ,..--* ~--

0.00 9 ... 0 0 30 60 90 120 150

AUTOLYSIS TIME (MIN)

Figure 8 Release of ConA-reactive teichoic acid from cell walls of B. subtitis 168. Autolysis was carried out at room temperature in 40 mM Tris-HCl (pH 7 .4). As autolysis proceeded, samples were withdrawn, heat inactivated at 100°C for 15 min, and mixed with radioactive ConA (final volumes were 2.0 ml containing 2.0 mg ConA of 1940 cpm/mg). After an incubation, ConA-teichoic acid or autolysate complexes were removed by centrifugation, washed, and radioactivity determined on supernatant fractions. Wall concentration was 0.5 mg/ml. (From Ref. 75.)

32 Doyle

Figure 9 Binding of fluorescein-ConA to B. subtilis gta strain C33 after a shift from permissive (35°C) to nonpermissive (45°C) conditions. Intervals (generations) after the shift: 0 (A), 1.6 (B), 2.8 (C), 3.2 (D) and 5.0 (E). Growth rates were 33 min per generation at the nonpermissive temperature. (From Ref. 40.)


Figure 10 Interaction between tluorescein-ConA and B. subtilis gtaC33 after a shift from nonpermissive to permissive conditions. Cells were prepared for photog-raphy 0.8 generations after the temperature shift. Note that the cell poles are not as fluorescent as the cell cylinders or the septa. Growth rate in the minimal medium was 40 min per generation at the permissive temperature. Cells grown at the nonper-missive temperature were unable to bind the lectin. Bar, 15 1-1m. (From Ref. 40.)

membrane, followed by even more addition of wall. New wall pushed old wall away from the cytoplasmic side to the cell surface. More recent refine-ments of the mechanisms of surface expansion of bacilli have been pre-sented by Kirchner et al. [148] and by Kemper et al. [149]. The results provide a good description of how a lectin (ConA) has been used to study a major problem in bacterial physiology. Figure 11 shows that as the temper-ature-sensitive bacilli are shifted from permissive to nonpermissive condi-tions, nearly three generations are required before most ConA-reactive sites are turned over or diluted. Presumably, these ConA-binding sites are resid-ual side wall materials and old poles. In contrast, when the cells are shifted from nonpermissive conditions, only one generation is needed for ConA-reactive sites to appear. The newly inserted sites are from new poles (septa) and side wall material "pushed" to the outer surface.

34 Doyle

a: 260 0 u.. o_ 35° ~ 45° 45° ~ 35° w-a:.€ -o ::J:t 180 o-wz a:o <-

~ z(!) 120 -'w

~a: <((!) Z(!) << {) z 40 0 {)

2 3 4 5 2 3 4

GENERATIONS

Figure 11 Agglutination of B. subtilis gtaC33 by ConA. The panel on the left shows the amount of ConA required to aggregate the cells when the growth tempera-ture was shifted from a permissive to a nonpermissive condition. Cells were re-moved, mixed with ConA and examined for aggregation under a dissecting micro-scope. The panel on the right shows the aggregation results when the cells were shifted from a nonpermissive to permissive temperature. (Some of the results cour-tesy ofHLT Mobley.)

XI. MICROBIAL ULTRASTRUCTURE AS PROBED BY LECTINS

Concanavalin A has also been employed to study the organization of tei-choic acid in the wall of B. subtilis 168 [150]. Thin sections of the walls of the bacterium were relatively uniform, but ConA-bound walls exhibited an asymmetry (Figs. 12 and 13). The ConA-treated walls exhibited irregular fuzzy external surfaces, whereas untreated walls were relatively smooth. It was speculated that the lectin condensed the peripheral teichoic acids, re-sulting in the observed asymmetry of the walls (Fig. 14). The current view is that the outer surface of the walls are serrated because of cellular turgor and autolysins [149].

In Staph. aureus, walls also seem to be serrated or rough on the outer surface. Morioka et al. [148] observed that colloidal gold-WGA labeled partially autolyzed cells very densely (Fig. 15). Wall material could be ob-served sloughing off from the cell surface. The sloughed off wall material


Figure 12 Thin sections of B. subtilis 168. (a) Untreated; (b) treated with ConA. (From Ref. 150.)

is, no doubt, due to turnover of wall during normal cell surface expansion [92]. Figure 15 shows the labeling pattern of WGA-gold on S. aureus. Morioka et al. [151] considered that the WGA was binding to cell wall teichoic acids. They did not show micrographs of isolated walls, but be-cause of staining of thin sections postembedding, were able to show the binding pattern of the lectin on the inner- and outer-wall faces. Interest-ingly, the WGA seemed to bind to three distinct layers in the septum or

36 Doyle

Figure 13 Thin sections of cell walls of B. subtilis 168. (a,b) untreated controls; (c,d) treated with ConA. (From Ref. 150.)

cross-wall. When staining with WGA-gold was done in the presence of GlcNAc there was no evidence of labeling.

Electron microscopic techniques with lectin probes have been used to study the surface localization of lipoteichoic acids in group A streptococci [152]. Concanavalin A labeled with ferritin was employed to show that the lipoteichoic acid (L T A) could be found on the outer surface of the cell. Pepsin digestion of the cells resulted in increased binding of ConA-ferritin. Furthermore, isolated walls were also capable of binding the lectin, but


only on the periphery of the walls. Strains exhibiting high hydrophobicities bound more lectin than the hydrophilic strains. The results are consistent with the view that LT A molecules are surface-exposed in group A strepto-cocci and may serve in interacting with mucosal cells. The specificity of ConA binding to cellular (and wall) LT A was confirmed by first mixing the lectin with purified L T A. The purified L T A caused a reduction in the binding of ConA to cell surfaces. The results are interesting from the view-point that a membrane-anchored L T A molecule can become exteriorized and bind strongly to cell walls. The walls must have complementary recep-tors for the L T A molecule. The addition of L T A to L T A-depleted walls was not attempted, although such experiments may be useful in establishing the identity of LT A receptors on cell walls. The ConA-ferritin (and gold) probes employed [152] demonstrate the value of lectins in characterizing microbial surface adhesins.

Maruyama et al. [101] isolated two mutants of E. coli that were highly sensitive to sodium dodecyl sulfate (SDS). Both of these mutants were of

Untreated Cell Walls

I 30 ••

Cell Walls fallowing Fixation and Dehydration

41 nm

25 nm

Surface Teichoic Acid Stainable Cell Wall

Cell Walls plus Concanavalin A

Concanavalin A

• • • • • • • • • • • • • • • • • • Figure 14 Schematic representation of the organization of the cell surface of B. subtilis 168. (From Ref. 150.)

38 Doyle

Figure 15 Binding of wheat germ agglutinin to cell walls of Staphylococcus aureus. (A) Thin section of Staph. aureus incubated with WGA-gold. Label is found on both the inner and outer wall faces. (B) Same as A, but the label is shown to bind strongly to a septum. (C) Interaction of WGA-gold with a partially autolyzed Staph. aureus. (From Ref. 151.)

readily aggregated by ConA, but the parent isogenic strain was relatively refractory to the lectin. Presumably, the mutants contained defective outer membrane components, leading to exposure of glycoconjugates on the plasma membrane. Maruyama [153-155] employed the agglutinability by ConA to assay for spheroplast formation in Escherichia coli. The identity


the glycoconjugate(s) on the spheroplast surface capable of interacting with ConA has not been defined, although lipopolysaccharide structures may be involved.

Lectins have been employed to study the surface carbohydrates in vari-ous morphological forms of rumen fungi [156]. For example, the fucose-specific Laccaria amesthystina lectin bound spores, flagella, sporangia, and rhizoids of a Neocallimastix strain, but in another strain, the flagella were unable to bind the lectin. Similarly, the same lectin bound to the rhizoids of some Piromonas strains, but not to others. Lectins not only distinguished between various morphological structures, but also distinguished the genera Neocallimastix, Piromonas, and Sphaeromonas [156]. The lectin-binding sites were located on the fungal structures by fluorescence. Lectins thus serve as powerful probes to study the surface differentiation of life cycles of fungi.

Bonfante-Fasolo et al. [157] were able to use WGA-gold to localize chitin in the spore walls of the fungus Glomus versiforme. Chitin was localized in the fibrillar wall components. Labeling was not observed in the cytoplasm or the areas separating primary and secondary wall. The ultrastructural results are in agreement with the notion that chitin synthesis occurs at the plasmalemma level. Later studies by these authors [158], employing lectin-gold markers, were directed to determining the surface location of carbohydrates in the fungal symbiont, Hymenoscyphus ericae. They showed that WGA-gold bound exclusively to septa and to an inner electron transparent layer on longitudinal walls. The ConA-reactive mate-rial was seen to be radiating from the wall of infective strains, suggesting that this material was involved in adhesion to host cells. In fact, when the infective strain was in contact with the host, there was evidence for an abundance of ConA-gold sites. The results may be useful in establishing the molecular basis for fungi-plant interactions.

Miragall et al. [159] and Rico et al. [160] have studied the surface expansion in Candida albicans, an opportunistic fungal pathogen. They observed that protoplasts of C. albicans lacked WGA-gold-reactive sites (or WGA-peroxidase sites), but when the protoplasts were allowed to re-generate their wall materials, the WGA sites were synthesized slowly. The initial WGA-reactive sites were found on or near the plasmalemma, but later sites were marked rather uniformly over the entire surface. The ConA-ferritin sites appeared on the very outermost wall layer as the protoplasts were converted into vegetative cells. Although the ConA sites were consid-ered to be synthesized only during later stages of division, there is no obvious explanation of how the sites are assembled on the surface. Rico et al. [160] established that a lytic process in localized areas of the wall was required for surface expansion. This process may result in cell wall turnover [92], but this is not known with certainty. It is thought that yeasts add their

40 Doyle

newly synthesized materials at sites of septation and at diffuse sites covering the growing bud.

Concanavalin A is a marker for the mannoproteins of yeasts, including C. albicans [31,37 ,94]. From the results of Miragall et al. [159] and Rico et al. [160], it seems that the assembly of yeast cell wall occurs in distinct steps. The initial steps involve the synthesis of chitin (WGA-reactive), whereas the latter steps incorporate mannoproteins (ConA-reactive). This is one exam-ple of the application of lectins in the study of microbial growth processes.

XII. INFLUENCE OF LECTINS ON MICROBIAL PHYSIOLOGY In an interesting application of lectins in microbial physiology, it was ob-served that ConA prevented the uptake of DNA by B. subtilis [161]. Compe-tent cultures of B. subtilis were mixed with the lectin and transforming DNA added. The ConA prevented the expression of new genetic markers, but the effect of ConA was negated by methyl-a-n-glucoside. In control experi-ments, it was established that the lectin had no effect on cell viability. It ap-pears that ConA sequestered potential DNA receptors (probably teichoic acids) on the cell surface, rendering the cells incapable of binding the DNA.

Although ConA is unable to aggregate B. cereus 14579, the lectin can induce several physiological responses in the organism. For example, ConA stimulated growth rates and culture yields of the bacterium [162-164]. In addition, it increased oxygen uptake, and RNA, DNA, and protein synthe-sis in cultures of B. cereus. The enhanced physiological responses were unrelated to the lectin serving as a source of amino acids, because methyl-a-o-mannoside reversed the effects of ConA and, in control experiments, it was shown that the lectin was not internalized. Cell fractionations revealed that ConA was capable of binding the cytoplasmic membrane, but not the cell wall. These experiments are analogous to the now familiar studies on ConA-induced mitogenesis of lymphocytes. There is no obvious explana-tion for the effects of ConA on B. cereus. The authors speculate the cyclic guanosine 3 ',5 '-monophosphate (cGMP), also increased in the presence of the lectin, may regulate macromolecular synthesis. As far as is known, these studies have not been followed up. Additional studies are needed to define the mechanism of how ConA triggers physiological responses in bacilli. Lectin-enhanced metabolism could be of significant value in indus-trial microbiology.

XIII. LECTIN-BINDING PROTEINS OF BACTERIA Only a few bacteria are able to glycosylate their proteins [165]. The most widely studied glycoproteins of bacteria belong to the Archaeobacteria, although a few eubacteria have been reported to synthesize carbohydrate-


conjugated proteins. Bose et al. [166] reported that purified elementary bodies of the pathogen, Chlamydia trachomatis, were unable to bind to HeLa cultures in the presence of WGA. Later, Swanson and Kuo [167,168] were able to show that C. trachomatis proteins could bind to ConA, DBA (Dolichos biflorus), Ulex europaeus (Ulex-1), SBA (soybean agglutinin), and PNA (peanut agglutinin). The results were consistent with the bac-terium having lectin receptors consisting of mannose, fucose, and N-acetylgalactosamine (or galactose). Two proteins of 18 and 32 kDa were isolated, and when treated with periodate, were unable to bind the lectins. Furthermore, the two polypeptides were able to bind to the surfaces of HeLa cells, suggesting that they were adhesins. These proteins may be candidates for a chlamydial vaccine.

Mycoplasma pneumoniae, a wall-free bacterium, has been reported to contain glycoprotein in its plasma membrane [169]. These glycoproteins may be the receptors for lectins known to selectively interact with mycoplas-mal membranes [90]. Kahane and Tully [90] found that WGA, RCA, and ConA would bind to cells of Mycoplasma and to plasma membranes, al-though the binding of WGA was low. Proteolysis of the membranes led to an increase in lectin-reactive sites, suggesting that the carbohydrates may be partially masked in vegetative cells. In addition, the lectins tended to complex only the outer surface, showing that carbohydrate distribution in the membranes was asymmetric. Later, Kahane and Brunner [169] isolated a glycoprotein with a relative molecular mass (Mr) of 60,000 from M. pneumoniae membranes. The glycoprotein contained about 70Jo of weight of carbohydrate (Ole, Gal, GleN). No studies on the linkage of carbohy-drate to peptide or carbohydrate to carbohydrate were performed, so it is not proved that the lectin receptors were indeed the 60,000 Mr protein. Carbohydrates on bacteria may be recognized by macrophage surface lee-tins, leading to lectinophagocytosis [56]. Whether the binding of carbohy-drates by macrophage lectins contributes to the pathogenesis of the organ-ism is as yet unknown.

A WELLA technique was used to detect a glycoprotein in the plasma membrane of the mollicute, Spiroplasma citra [170]. Membranes were ex-tracted with chloroform/methanol to remove lipids and then fractionat-ed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The proteins were blotted onto nitrocellulose membrane filters, which were then overlaid with [3H]ConA. Autoradiography revealed the presence of a ConA-binding protein of about 84,000 Mr. Affinity chroma-tography on ConA-agarose was then used to isolate the glycoprotein. Cur-rently, no function has been ascribed to the glycoprotein. Furthermore, the nature of the linkage(s) between the polypeptide and its glycan substitu-ent(s) remains to be defined.

42 Doyle

A larvacidal toxin from spores of Bacillus sphaericus contains 120Jo carbohydrate, but cannot be purified by ConA chromatography [171]. Lec-tin probes have been employed to rule out the presence of carbohydrate on a surface array protein of Campylobacter fetus [172]. Many surface array proteins studied to date have been glycoproteins. The C. fetus surface array protein, although highly hydrophobic, would not bind to ConA [172]. An interesting glycoprotein autolysin was isolated by Kawamura and Shock-man [173] with the aid of ConA-Sepharose chromatography. The autolysin was glucosylated, a rare finding for glycoproteins. It seems unlikely that glucosylation was by a nonenzymatic reaction, because subfractions of the autolysin contained oligomeric glucose residues.

Further studies on possible glycoproteins in prokaryotes have been provided by Aitchison et al. [93]. They studied surface antigens of Strep. faecalis by electrophoresing whole-cell extracts, cell walls, and other cell fractions. Following SDS-PAGE, the gels were blotted onto nitrocellulose papers and mixed with lectin-peroxidase conjugates (WELLA). It was ob-served that lectins specific for L-fucose (UEA), D-glucose to D-mannose (ConA) and N-acetylglucosamine (WGA) bound to two prominent protein bands. Proper controls were run to eliminate the contributions of growth medium constituents. The results suggest that Strep. faecalis elaborates glycoprotein antigens. The attachment of the carbohydrates to polypeptide has not been studied, however. From the reports of Kawamura and Shock-man [173] and of Aitchison et al. [93], it seems that glycoproteins may be more common in prokaryotes than heretofore recognized.

XIV. THE GRAM STAIN AND LECTINS

Sizemore et al. [132] proposed that WGA could be used to selectively bind gram-positive cells, whereas the gram-negative bacterium would fail to in-teract with the lectin. They found that all gram-positive cells tested bound fluorescein-labeled WGA. Only some Pseudomonas sp. of the gram-negative bacteria yielded binding reactions with WGA. This study, although detailed in its testing of a variety of gram-positive and gram-negative bacteria, must account for some important exceptions. For example, many members of the genus Bacillus do not bind the WGA [76]. Furthermore, the gram-negative N. gonorrhoeae readily complexes with WGA [104]. Members of the genera Brucella and Yersinia have also been reported to interact with WGA [174,175]. Gram-negative bacteria with defective outer membrane, or with lipopolysaccharides containing !3-1,4-linked GlcNAc may be ex-pected to interact with WGA. It seems unlikely that the proposed test will be adopted; however, it is possible that newly discovered lectins will be able to discriminate between gram-positive and gram-negative bacteria.


APPENDIX

Sources and Specificities of Common Lectins

Systematic name Common name or source Specificities

A aptos papillata Sponge (AAP) (lsolectins) (GleNAc/3-1,4)n Abramis brama Fish (ABD) L-Rha >a-D-Gal

(Anti-B) Abrus precatorius Jequirty bean (ABP) /3-o-Gal > GleNAc Achatina fulica Snail (ACF) o-Gal Achatina granulata Snail (ACG) Sialic acid Adenia digitata Shrub (ADD) o-Gal Adenia volkensii Shrub (ADV) /3-D-Gal Aegopodium podagraria Ground elder (AEP) o-GalNAc Agaricus bisporus Common mushroom (AGP) Gal/3-1 ,3GalNAc;

Gal Agardhiella tenera Red alga (AGT) Complex Agaricus campestris Fungus (AGC) Complex Agrocybe aegerita Mushroom (AGEE) Complex Agropyrum repens Couch or quack grass (AGR) Gal/3-1 ,3GalNAc;

GalNAc (Anti-A) Albizzia julibrissin Mimosa tree seed (ALJ) Complex Alcyonium digitatum Marine cnidarium (ALD) a- or /3-o-Gal Aleuria aurantia Orange peel fungus (ALA) a-L-Fuc; a-L-Fuc a-

1,2Gal Allium cepa Onion (ALCE) o-Man; o-Gle Allium porrum Leek (ALPO) o-Man; o-Gle Allium sativum Garlic (ALS) Complex Allomyrina dichotoma Japanese beetle (AlloA) o-Gal/3-1 ,4GicNAc Aloe arborescens Aloe plant (Aloe) Complex Amaranthus caudatus Inca wheat (AMA) o-Gal Amaranthus leucocarpus Tropical herb (AML) Gal/3-1 ,3GalNAc

[Anti-M(T)] Amaranthus retroflexus Pigweed (AMR) Ole, Man Amaroucium stellatum Tunicate (AMS) Complex Amphicarpaea bracteata Hog peanut (AMB) GalNAca-

1,3GalNAc (Anti-A1)

Amphicarpeae edgeworthy Hog Peanut (AME) Complex (Anti A>H)

Androctonus australis Scorpion (ANA) Sialic acid Angelica archangelica Medicinal plant (ANA) Glycoprotein Anguilla anguilla Eel (AAA) L-Fuca-1 ,3Gal [Anti-

O(H)] Anguilla rostrata Eel (ANR) L-Fuc

44 Doyle


Anopheles albopictus Mosquito (ANAL) Complex Antheraea pernyi Chinese oak silk moth (ANP) a({j)o-Gal Anthocidaris crassispina Sea urchin (eichinoidin) Gal{j-1 ,3GalNAc Antirrhinum majus Snapdragon (ANM) Glycoprotein Aplysia californica Sea hare (APC) Complex Aplysia depilans Sea mollusc (APD) o-Galacturonic acid;

D-Gal Arachis hypogaea Peanut (PNA) Gal{j-1 ,3GalNAc;

Gal (Anti-T1Tt, Th, TJ

Arion empiricorum Snail (ARE) Complex Aristolachia galeata Medicinal herb (ARG) D-Gal (Anti-B>A) Artocarpus altilis Breadfruit (ARA) Gal[j-1 ,3GalNAca

(Anti-T) Artocarpus heterophyllus Jackfruit Gacalin) (JCA) Gal{j-1 ,3GalNAc

(integrifolia) (Anti-T) Artocarpus lakoocha Jackfruit (ATL) Gal[j-1,3GalNAc;

a-D-Gal Ascidia malaca Sea squirt (ASM) Gala-1,6Glc>Gal Asterias forbesi Sea star (ASF) Complex Astragalus onobrychis Milk vetch (ASO) D-GalNAc (Anti-A)

(distortus) A vena sativa Oat(OAT) [j-o-Glc Axinella polypoides Sponge (AXP-1, II) (lsolec- Gal[j-1 ,6Gal

tins) Azolla caroliniana Water fern (AZC) D-Gal Balea perversa Snail (BAPE) D-Gal (Anti-B,A) Bauhinia candicans Shrub (BAC) D-Gal; GalNAc Bauhinia carronii Shrub (BACA) [j-D-Gal Bauhinia purpurea Camel's foot tree (BAP) Gal[j-1,3GalNAc;

Gal{j-1,3Gal (Anti-T)

Bauhinia tomentosa Shrub (BAT) Complex Beta vulgaris Beet root (BEV) Complex Biomphalaria glabrata Water snail (BIG) (Isolectins) Fru > L-Gal >o-Man

(Anti-A1 > A2 >B) Birgus latro Coconut crab (BIL) Sialic acid Boltenia ovipera Tunicate (BOO) Sialic acid Boodlea coacta Green alga (BOC) (lsolectins) Glycoprotein Botylloides leachii Colonial asidian (BOL) D-Gal

(Isolectins) Bowringia milbraedii Shrub (BOM) o-Man Brachypodium sylvaticum Brome grass (BRS) (GlcNAc[j-1 ,4)n

> >GlcNAc



Brussica oleracea Red cabbage (BRO) Complex Bryonica dioica White bryony (BRD) GalNAc;Gala-1 ,6Gal Bryopsis hypnoides Algae (BRH) o-Gal [Anti-

B>A(H)] Buteo frondosa Bastard teak (BUF) Fuca1 ,2Gal;

Gal{j-1 ,3GlcNAc Calendula officina/is Artichoke or pot marigold o-Gle; Man

(CAO) Callinectes sapidus Blue crab (CAS) Complex Calliphora erythrocepha/a Blowfly (CAE) o-Man; o-Gle Cu/purina aurea (CAA) o-Gal;GalNAc (Anti-

A,B) Canavalia ensiformis Jack bean (Con A) Mana-1;Glca-1;

GlcNAca-1 Canavalia gladiata Japanese jack bean (Con G) Mana;Glca Cancer antennarius California or blue crab (CAA) Sialic acid Canna indica Indian herb (CAl) Hydrophobic 13-

glucosides Capsicum annuum Hot herb (CAA) ( GlcNAc{j-1 ,4)0

Caragana arborescens Siberian pea tree (CAAR) o-GalNAc (lsolectins)

Caragna frutex Siberian pea shrub (CAFR) o-Gal [Anti-B>A(H)]

Carcinoscorpius rotunda Horseshoe crab (Carcino- Sialic acid cauda scorpin) (NeuAca-2,6Gal)

Carpopeltis flabellata Marine alga (CAF) Glycoproteins Caucasotachea astro- Snail (CAAS) o-GalNAc (Anti

/abiata A>H) Caulerpa paspa/oides Marine alga (CAP A) Complex Cepaea hortensis Snail (CEH) Sialic acid Cepaea nemora/is Snail (CEN) o-GalNAc (Anti-

A>H) Cerastium tomentosum Snow in summer (CET) Complex Cercis siliquastrum Red bud or Judas tree (CES) Complex Chamaespartium sagittale Winged broom (CHS) Complex Channa leucopunctatus Fish (CHL) (Isolectins I-III) GalNAca-

1,3GalNAc; GalNAc

Charybdis japonica Crab (CHJ) o-Gal (Anti-B >A) Chelidonium majus Greater celandine (CHM) o-GlcNAc Cicer arietinum Chick pea (CIA) Complex Citrullus vulgaris Watermelon (CIV) Man;Glc;GlcNAc Cladonia pyxidata Marine lichen (CLP) Glc;Man (Anti

A>AB)

46 Doyle


Clave/ina picta Tunicate (CLPI) Complex Clerodendron inerme Asian shrub (CLI) Complex Clerodendron trichotomum Asian shrub (CL T) o-GalNAc;Gal Clitocybe geotropa Fleshy mushroom (CLG) a-L-Fuc Clitocybe nebularis Nebelkappe (CLN) o-GalNAc;Gal Clupea harengus Herring egg (CLHO) o-Rha,o-Gal (Anti-

B>A1)

Codium fragile Sponge seaweed (COF) o-GalNAc (Anti-A) Colchicum autumnale Meadow saffron (COA) a,{l-o-Gal;GalNAc Colinus coggygria Cashew like shrub (COC) Glycoprotein Colocasia esculenta Taro (COE) (lsolectins) Complex Coronilla varia Crown vetch (COY) o-Gal;GalNAc (Anti-

A>B) Cotinus ceggyaria Smoketree (COCE) Complex. Crassostrea virginica Oyster (CRY) Complex Crenomytilus grayanus Mussel (CRG) o-Gal Crotolaria aegypteaca Middle East shrub (CRA) o-GalNac (Anti

A>H) Crotolaria juncea Sunhemp (CRJ) o-Gal > GalNAc Crotolaria striata Shrub (crotalarin) o-GalNAc > o-Gal

(Anti-A) Croton tiglium (Euphorbiaceae) (CRT) Complex Cucumis sativa Cucumber (CUS) Glycoprotein Cucurbita maxima Winter squash (CUM) (GlcNAc{l-1,4). Cucurbita pepo Squash (CUP) (GlcNAc{l-1 ,4).;

GlcNAc Cuscuta europea (gronovii) Dodder (CUE) Complex Cycad siamensis Primitive gymnosperm (CYS) Complex Cynara scolymus Globe artichoke (CYSC) Glycoprotein Cystoclonium purpureum Marine alga (CYP) Complex Cytisus (Sarothamnus) Scotch broom (CYSCO) o-Gal;o-GalNAc

scoparius Cytisus sessilifolius Shrub (CYSE) (lsolectins) (GlcNAc{l-1,4).;

cellobiose; L-Fuca-1,2Gal [Anti O(H), A2]

Datura innoxia Harmless jimsonweed (DAI) (GlcNAc{l-1 ,4). Datura stramonium Thorn apple (DAS) Gal{l-1,3(4)GlcNAc

(or Jimsonweed) Daucus carota Carrot (DAC) (GlcNAc{l-1,4). Dicentrarchus, labrax Sea bass (DIL) o-Fuc (Anti-H) Dictyostelium Slime mold (discoidin) (lso- o-GalNAc;o-Fuc

discoideum lectins)



Dictyota dichotoma Brown alga (DID) Complex Didemnum candidum Ascidian (DIC-1,11) Gal(p-1,4)Fru;

(lsolectins) Galj3-1,4Glc;o-Gal Dioclea grandijlora (similar to jack bean) o-Man >o-Gle

(DIG) Dolichos bijlorus Horse gram (DBA) GalNAca-

1,3GalNAc (Anti-A1 > A 2 >Cad)

Drosera rotundifolia Sun dew (DRR) Complex Echinocereus engelmanii Cactus (ECE) Complex Electrophorus electricus Electric eel (EEL) o-Gal Elettaria cardamomun Cardomon (ELC) Complex Eobania vermicu/ata Snail (EOV) o-GalNAc (Anti-

A>H) Eranthis hyemalis Winter aconite root (ERH) Galj3-1,4GlcNAc;

Galj3-1,4Glc (Anti-O(H)>A,B)

Erythrina corrallodendron Coral tree (ECor) Galj3-1,4GlcNAc; GlcNAc > o-Gal

Erythrina cristagalli Coral tree (ECA) Galj3-1,4GlcNAc; Gala 1,6Gal;Gal

Erythrina variegata Coral tree (EVA) Galj3-1,4GlcNAc Euhadra cal/izona Snail (EUC) Complex

(amaliae) Euhadra periomphala Snail (EUP) o-GalNAc (Anti-

A>H) Euphorbia antiquorum Evergreen (EUA) o-Gal Euphorbia cyparissias Cypress spurge (EUC) a-D-Gal Euphorbia heterophylla Evergreen (EUH) o-GalNAc >Lac Evonymus europaeus Spindle tree (EUE) Gala-1,3Gal (Anti-B) Fagopyrum esculentum Buckwheat (FAE) Complex Falcata japonica Shrub (FAJ) o-GalNAc;Gal (Anti-

AI>A2) Fornes fomentarius Sapwood rot (FOF) a-o-Gal;GalNAc

(Anti-B>O) Fucus vesiculosus Brown alga (FUV) Complex Galanthus niva/is Snow drop (GAN) Mana-1,3Glc Galega officina/is Goat's rue (GAO) o-Glc;Man Ganoderma /ucidum Fairies teaspoon (GAL) !3-o-Gal

(applanatum) Geodia cydonium Sponge (GEC) Galj3-1,4GlcNAc;

Galj3-1,3GlcNAc; Gal;GalNAc

Genista tinctoria Greenweed (GET) Complex

48

Systematic name

Geum urbanum Glossina fuscipes

Glycine max (soja)

Gracilaria tikvahiae (confervoides)

Griffithsia flocculosa

Griffonia simplicifolia

Grifola frondosa

Gypsophila elegans Halichondric panicea

Halocynthia roretzi Hardenbergia comptoniana Helianthus annus Helix aspersa

Helix hortensis Helix lucorum Helix pomatia

Common name or source

Town herb (GEU) Tsetse fly (TSF) (numerous

other Glossina spp also pro-duce lectins)

Soybean (SBA) (lsolectins) I

Doyle

Specificities

Glycoprotein Complex

GalNAca(orml,3 Gal;o-Gal

Ceylon moss (GRT)

II 4-0-Methyl-D-glucuronic acid

Sialic Acid (Anti-A,B)

Red alga (GRF)

African legume (lsolectins)

GS-1

GS-IB4

GS-IAB3

GS-11

GS-IV

Mushroom (GRFR) (lsolectins) Maiden's breath (GYE) Marine sponge (AAP)

Solitary ascidian (HAR) Shrub (HAC) Sunflower (HEA) Garden snail (HEAS)

Snail (HEH) Snail (HEL) Garden snail (HEP)

(I) (II)

Complex (activity en-hanced by GalNAc or GlcNAc)

a-o-Gal;o-GalNAc (Anti-A,B)

a-D-Gal (Anti-B) a-o-Gal; o-GalNAc

(Anti-A,B) a-o-Gal;o-GalNAc

(Anti-A,B) a-o-Gal;o-GalNAc

(Anti-A,B) a-o-GalNAc

(Anti-A) o-GlcNAc (Anti-

B,Tk,T) a-L-Fuc-1,2Gal

(Anti-Leb andY) o-Gal > GalNAc o-GalNAc Complex L-Fuc; o-GlcU;

o-GalU oGal; Mel Raf Complex a-o-GalNAc

(Anti-A) Sialic acid a,(j-GalNAc GalNAca-

1,3GalNAc (Anti-A)



Helleborus purpurascens Hellebore (purple bear's foot) Complex (HEP)

Hevea brasiliensis Rubber tree (HEB) GlcNAc(l3-1 ,4). Hippeastrum spp Amaryllis (HIA) Mano:-1 ,3Gal;Mano: Homarus americanus Lobster (HOA) (L-Ag1) Sialic acid

(lsolectins) (L-Ag2) o-GalNAc Hordeum vulgare Barley (HOV) GlcNAc Hura crepitans Sand box tree (HUC) o-GalNAc >Gal Hypnea japonica Marine alga (HYJ) (lsolectins) Glycoproteins Hyptis suaveolens Tropical tree (HYS) Gal;GalNAc

(Anti-A) lberis amara Shrub (IBA) Complex [Anti-

M(M+N)) lberis umbellata Shrub (IBU) 0-Gal lphygena plicatu/a Snail (IPP) o-Gal;Rha [Anti-

B,A(H)] Ipomoea rubrocoerulea Morning glory (IPR) o-Glu;Man

(purpurea) Ischnoderma resinosum Mushroom (ISR) 13-o-Gal (Anti-A) Laburnum a/pinum Golden chain (LAA) L-Fuco:-1 ,2Gal

[Anti-O(H)] Laburnum anagyroides Bark (LBA) L-Fuc [Anti-O(H)] Laccaria amethystina Mushroom (Isolectins) 13-o-Gal;o-GalNAc

(LAAM) L-Fuc [Anti-O(H)] Lactarius deliciosus Mushroom (milky cap) (LAD) Gall3-1 ,3GalNAc Lactarius perlatum Red pepper (LAP) 13-o-Gal Laelia autumnalis Orchid (LAAU) o:,l3-o-Gal (Anti-

A1>A2 >Le•> Leb)

Lathyrus ochrus Vetchling (lsolectins) (LoL I, o-Man;o-Glc II)

Lathyrus odoratus Sweet pea (LAO) o-Man> o-Gle> GlcNAc

Lathyrus sphaericus Pea (LAS) o-Man;Glc Lathyrus sylvestrus Purple sweet pea (LASY) o-GalNAc (Anti-

A>H) Laurencia undulata Algae (LAU) o-Gal (Anti-

B>A>H) Laurus nobilis Bay leaf (LAN) Complex Leciniaria biplicata Snail (LEB) o-Gal,Rha [Anti-

B,A(H)] Lens culinaris (esculenta) Lentil (LCA) Mano:-1 ;Glco:-1 ;NAc Leonurus cardiaca Motherwort (LEC) 13-o-GalNAc (Anti-

Cad, but not Tn)

50 Doyle


Lepidium sativum Garden cress (LES) Complex Leucojum vernum White snowflower (LEV) o-Man Levisticum officinale Aromatic herb (LEO) Complex Limax flavus Slug (LFA) Sialic acid Limulus polyphemus Horseshoe crab (limulin) (LIP) Sialic acid Listera ovata Twayblade (LIO) o-Man Litchi chinensis Lychee nut (LIC) Complex Lonchocarpus capassa Apple leaf (LOC) Complex Lophocereus shotti Cactus (LOS) Complex Lotononis bainesii Perennial pasture legume Complex

(LOB) Lotus tetragonolobus Asparagus (LOTUS) (winged L-Fuca-1 ,2Gal [Anti-

pea) O(H)] Luff a actangula Gourd (LUA) (GlcNAc{l-1 ,4). Lumbricus terrestris Earthworm (LUT) Complex Lycoperdon perlatum Puff ball mushroom (L YP) Complex Lycopersicon esculentum Tomato (LYE) (GlcNAc{l-1 ,4). Lygos monosperma Willow-like reed tree (L YM) Complex (Anti-Cad,

but not T, Tk, Th, T.)

Lygos sphaerocarpa Reed tree (L YS) Complex (Anti-T, T "' but not Cad)

Lyngbya majusarla Algae (L YMA) Complex Machaerocerus eruca Baja cactus (MAE) (lsolectins) a-L-Fuc;o-GalNAc Madura pomijera Osage orange (MAP) Gal{l-1 ,3GalNAc

(Anti-T) Macrotyloma axil/are Shrub (MAAX) o-GalNAc (Anti-

At>A2) Mangifera indica Mango (MAl) Complex Marasmius oreades Fairy-ring mushroom (MAO) o-Gal (Anti-B>O) Medicago lupulina Black medic (MEL) Glycoproteins Medicago sativa (truncu- Alfalfa (MES) o-Man;Glc

lata) Megabalanus rosa Barnacle (MER) (lsolectins) o-Gal Megapetaria squa/ida Clam (MES) o-GalNAc Microciona porifera Marine sponge (MIP) Complex Moluccella laevis Irish bell (MOL) Complex (Anti-A,N) Momordica charantia Bitter pear melon (MOC) o-Gal > GalNAc Moringia olifera Moringin Complex Myrica gale Sweet gale (MYG) Complex Narcissus pseudonarcissus Daffodil (NAP) Mana-1,3;Mana-1,6 Ocimum basilicum Basil (OCB) Complex Octopus vulgaris (bairdi) Octopus (OCV) {1-o-Gal



Oncorhynchus spp Salmon eggs a,{j-o-Gal Onobrychis viciifolia Sanfoin (ONV) a-Man> a-Ole Ononis hircinia Restharrow (ONH) a,{j-o-Gal;o-GalNAc Ononis spinosa Spiny restharrow (ONS) a-n-Gal Origanum vulgare Oregano (ORV) Complex Oryza sativa Rice (ORS) o-GlcNAc Otala lactea Snail (OTL) o-GalNAc (Anti-

A>H) Pachycereus pringlei Cactus (PAP) Complex (Anti-

A>B) Palmaria palmata Marine alga (PAP) Complex Papaver dubium Doubtful poppy (PAD) (GlcNAc{j-1,4)0

Papaver somnijerum Sleep-bringing or opium (GlcNAc(j-1 ,4)0

poppy (PAS) Pastinaca sativa Parsnip (P ASA) Complex Peltigera canina Lichen (PEC) Complex Pelvetia canaliculata Brown alga (PEC) Unknown Perea flaviatilis Perch (PEF) o-Glc;o-Man;L-Fuc Periplaneta americana Cockroach (PEAM) Complex Persea americana Avocado fruit seed (P AA) Complex Petromyzon marinus Lamprey (PEM) Complex Petroselinum crispum Parsley (PEC) Complex Phallus impudicus Stinkhorn mushroom (PHI) Complex Phanerochaete chryso- Fungus (PHC) (Glc(j-1 ,4)0

sporium Phaseolus acutiofolius Terpary bean (PHA) (lsolec- Complex

tins) Phaseolus coccineus Scarlet runner bean (PH C) Unknown Phaseolus lunatus (I, II) Lima bean (LBA) (lsolectins) GalNAca-1 ,3Gal

(Anti-A) Phaseolus vulgaris Red kidney bean (PHA) (lso- Complex

lectins) Gal(j-1,4 GlcNAc-(j-1,2Mana (Anti-A)

Phlebotomus papatasi Sandfly (PHP) (numerous Complex other Phlebotomus spp also produce lectins)

Phlomis fruticosa Jerusalem sage (PHF) Mel;GlcNAc; 2-deoxy-o-Gal

Phlox drumondii Ornamental thread (PHD) o-Glc;Man Phoradendrom califor- Desert mistletoe (PHCA) o-Gal

nicum Photo/iota squarrosa Broad-leaf tree (PHS) a-L-Fuc

52 Doyle


Phragmites australis Common reed (PHAU) (lso- o-GalNAc lectins)

Phytolacca americana Pokeweed (PWM) (lsolectins) Gal/3-1,4(3)GlcNAc; (GlcNAc,j3-1,4)0 ;

Mana-1,2Man Pichia anomala Yeast (PIA) (Glcj3-1,4)0

Pi/a globosa Snail (PIG) Sialic acid Pisum sativum Pea(PEA) Mana-1 ;Glca-1 Plecoglossus altivelis Fish egg (PLA) L-Rha (Anti-B) Pleurotus ostreatus Mushroom (PLO) Fucosyllactose [Anti-

O(H)] Pleurotus spodo/eucus Mushroom (PLS) Fucosyllactose [Anti-

O(H)] P/umaria elegans Marine alga (PLE) Complex Polyandrocarpa misa- Tunicate (POM) o-Gal

kiensis Polygonatum multiflorum Perennial flower, USSR Complex

(POMU) Po/ygonum persicaria Buckwheat or lady's thumb o-Gal

smartweed (POP) Psathyrel/a velutina Mushroom (PSV) o-GlcNAc Pseudomonas aeruginosa Bacterium (PA-l, PA-Il) Thiogalactosides

>o-Gal; L-Fuc;L-Gal; o-Man

Psophocarpus tetragono- Winged bean (PST) o-Gal > o-GalNAc lobus (Anti-A,B)

Pterocarpus ango/ensis South Afr. kiatt tree (PTA) o-Gle, o-Man Ptilota plumosa Red marine algae (PTP) o-Gal (Anti-B) Quercus rubra English or red oak (QUR) (GlcNAcj3-1,4)0

Rana catesbaiena Frog eggs (RAC) Complex, Sialic acid Rana japonica Frog (RAJ) Complex Raphanus sativus Radish (RAS) Glycoproteins Rheum rhabarbarum Garden rhubard (RHR) Glycoprotein Rhodnius prolixus Reduviid bug (RHP) o-ManNAc;

o-GalNAc; o-Gal

Ricinus communis Castor bean (lsolectins) Galj3-1,3(4)GlcNAc (RCA-1,11) Galj3-1,3GalNAc;

GalNAc Robinia pseudoacacia Black locust (ROP) Complex Rumex obtrisfolia Bitterdock (RUD) Hydrophobic 13-

glycosides



Rumex patientia Shrub (RUP) Glycoprotein Rutilis rutilus Fish egg a-L-Rha > {3-o-Gal Saccharomyces cerevisiae Yeast (SAC) o-Man Salix alba White willow (SAA) (GlcNAc{3-l ,4). Salmo solar Salmon (SAS) o-Gal (Anti-B>A) Salvia horminum Bluebeard shrub (SAH) Complex (Anti-

T.+Cad) Salvia sclarea Clary shrub (SAC) GalNAca-1 ,ser

(or thr) (Anti-T.) Sambucus edulus Daneworth (SAE) o-Gal (Anti-A> B,O) Sambucus nigra Elderberry (SNA) (lsolectins o-Gai;GalNAc;

1-11) Sialic acid Sambucus racemosa Shrub (SAR) Complex Sarcophaga peregrina Flesh fly (SAP) Complex Satureja hortensia Savory herb (SAH) Complex Saxidomus giganteus Butter clam (SAG) o-GalNAc (Anti-A,) Saxidomus purpuratus Shellfish (SAPU) (lsolectins) a-o-GlcNAc Sclerotium rolfsii Fungus (SCR) (lsolectins) Glc{3-1 ,3Glc Secale cereale Rye (SCL) o-GlcNAc Sesamum indicum Sesame (SEI) o-GlcNAc Simulium ornatum Blackfly (SIO) Complex Soja hipspida Bean (SOH) o-GlcNAc;Gal Solanum alatum Winged nightshade (SOA) (GlcNAc{3-1 ,4). Solanum dulcamara Woody nightshade (SOD) Complex Solanum melongena Eggplant (SOM) (GlcNAc{3-l ,4). Solanum tuberosum Potato (ST A) (GlcNAc{3-1 ,4)z_5

Solieria chorda/is Marine alga (SOC) Glycoprotein Sophora japonica Japanese pagoda tree (SOJ) Gal{3-1 ,3GalNAc;

Gal{3-l ,3GlcNAc [Anti-B>A> >

O(H)] Sorbus aucuparia European mountain ash Complex

(SDA) Spondyliosoma cantharus Sea bream (SPC) Complex (Anti

B>H) Streptomyces spp Bacterium (STR) a-L-Fuc;o-Man Stye/a plicata Tunicate (STP) Sialic acid Synadenium grantii Community seed tree (SYG) a-D-Gal Tachypleus tridentatus Asian horseshoe crab (TAT) Sialic acid Taxus bactata Gymnosperm or Yew (TAB) Hydrophobic

{3-glucosides Tetracarpidium cono- Nigerian walnut (TEC) Gal{3-1 ,4GlcNAc

phorum

54 Doyle


Tetragonolobus maritimus Winged pea (TEM) Glycoprotein Thymus vulgaris Thyme(THV) Complex Tichocarpus crinitus Red alga (TIC) Complex Tilia cordata Basswood (TICD) (GlcNAc/3-1 ,4)0

Trichosanthes anguina Gourd (hair flower) (TRA) Complex Trichosanthes kiri/owii Chinese gourd (TRK) D-Gal Tridacna crocea Mollusc (TRC) D-Gal Tridacna maxima Clam(TRM) Gal/3-1 ,4GlcNAc Trifolium repens White clover (Trifoliin) 2-Deoxyglucose Trigonella procumbens Fenugreek (TRP) Complex Trimeresurus mucrosqua- Poison scaly fungus (TRMU) Complex

matusvenum Triticum vulgaris Wheat(WGA) (GlcNAc/3-1,4)0 , Sia-

(aestivum) lie acid Tropaeolum majus Nasturtium (TRMM) D-GalNAc (Anti-A) Tu/ipa gesneriana Tulip (TUG) o-Man Ulex europaeus Gorse (or furze) (lsolectins) L-Fuca-1 ,2Gal[Anti-

(UEA-1, UEA-11) O(H)]; (GlcNAc/3-1 ,4)2 [Anti-O(H)]

Ulmus glabria Smooth elm (ULG) (GlcNAc/3-1 ,4)0

Ulva arasakei Algae(ULA) Complex [Anti-A,H(O)]

Ulva arasakii Algae (UAR) o-GalNAc (Anti-A+H)

Ulva lactuca Green marine algae (ULL) a-L-Fuc [Anti-O(H)] Urtica dioica Stinging nettle (URD) (GlcNAc/3-1 ,4)0

Vaejovis spinigerus Scorpion (VAS) Complex; sialic acids Velesunio ambigus Murray mussel (VV A) Complex Verbascum blattaria Smooth moth plant (VEB) Complex Viburnum lantana Wayfaring tree (VIL) Glycoprotein Viburnum opulus Guelder rose (VIO) Glycoprotein Vicia cracca Common vetch (VIC) o-GalNAc (Anti-A) Vicia cretica Vetch (VICRE) Complex (Anti-

TITh,A) Viciafaba Fava (broad) bean (favin) Mana-1;Glca-1 Vicia graminea Herb (VI G) Gal/3-1 ,3GalNAc

(Anti-N>O) Vicia hyrcania Shrub (VIH) (lsolectins) Gal/3-1 ;3GalNAc

(Anti-T) GlcNAc,Glc

(Anti-Tk) Vicia vi/losa Hairy vetch (VVA) (Isolectins) GalNAca-1,ser

(Anti-T0 ) (or thr)



Vigna radiata Mung bean (VIR) D-Gal Vigna ungiucu/ata Cowpea (VIU) D-Gal Viscum album Mistletoe (Isolectins) (ML-I, o-Gal; o-GalNH2

II, III) Wisteria jloribunda Japanese wisteria (WIF) GalNAca-1 ,6Gal;

GaiNAc;Gal Wisteria sinensis Flower (WIS) D-Gal Xenopus laevis Frog (XEL) a,/3-D-Gal Xylaria polymorpha Mushroom (XYP) Complex Zeamays Maize (ZEM) o-Man;Gal;GalNAc Zingiber officinale Chinese ginger (ZIO) Glycoprotein

Specificities determined by hemagglutination inhibition, equilibrium dialysis, fluorescence quenching, other. Specificities may reflect contributions of penultimate residues, or peptides. In some cases, specificities reported in the literature do not reveal anomeric preferences or blood group reactivities. In other cases, reports may disagree. When di- or tri-saccharide specificities are shown, usually the lectin has an affinity for the nonreducing carbohydrate residue. The table does not list hydrophobic derivatives of saccharides, although frequently the hydrophobic derivatives are capable of binding with higher affinities than unmodified saccharides. Many lectins have a higher affinity for di-, tri-, or multi-, antennary complex carbohydrates than for linear carbohydrate sequences, but the table has been simplifed to include only the latter. Vertebrate lectins, although now recognized to be numerous, are not thoroughly reviewed in the table. Finally, bacteriallectins, with the exception of Pseudomonas aeruginosa and Streptomyces spp, have not been purified in significant quantities and are not described in the table. Fru = fructose; Fuc = fucose; Gal = galactose; GalNAc = N-acetyl-galactosamine; Ga!NH2 = galactosamine; Ole = glucose, GlcNAc = N-acetylglucosamine; Lac = lactose; Man = mannose, Mel = melibiose; NeuAc = neuraminic acid, Raf = raffi-nose. Major commercial sources of lectins are E-Y Laboratories, San Mateo, CA (USA); Lectinola, Charles University, Prague, Czech Republic; Lectinotest, Lvov Medical Institute, Lvov, Ukraine and Sigma Chemical Company, St. Louis, MO (USA). Abbreviations are based on common usage and on first two letters of the genus and first letter of the species. In some cases, it is necessary to employ the first two letters of both the genus and species. Table was derived from the reviews of Mogos eta/. (61), Antonjuk eta/. (62), Sharon and Lis (9), Liener et a/. (8), Bird (20), Wu et al. (60), Etzler (40), Strosberg et a/. (63), Goldstein and Poretz (64), Goldstein and Hayes (59), Garber eta/. (66,67), Gilboa-Garber eta/. (65), and Mandai and Mandai (68), commercial pamphlets and original papers of numerous investigators.

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3

Epidemiological Applications of

Lectins to Agents of

Sexually Transmitted Diseases

WILLIAM 0. SCHALLA and STEPHEN A. MORSE Centers for Disease

Control and Prevention, Public Health Service, U.S.Department of Health and

Human Services, Atlanta, Georgia

I. INTRODUCTION

The first reported use of lectins to agglutinate bacteriawas published in

1936 and involved mycobacteria [1]; however, it was notuntil the 1960s

that lectins were used to study the cell surfacecarbohydrate moieties of

bacteria and fungi [2-5]. Since that time, the amount ofpublished informa

tion on lectin characterization of pathogenic andnonpathogenic microor

ganisms has dramatically increased. A review of theinteraction of bacteria

and fungi with lectins and lectinlike substances has beenpublished [6]. The

interested reader is referred to this as well as otherreviews [7-11] for further

information. Many studies on lectin-microorganisminteractions have focused on

lectin binding to whole organisms. Virtually anycarbohydrate-containing

cell surface structure has the potential of binding one ormore lectins.

Some of these components have been identified and includefungal cell

wall constituents, such as mannans [6,7] and chitin [6,7],and bacterial

components, such as capsular polysaccharides [6,7,12-14],lipopolysaccha

rides [6,7,12,13,15], teichoic acids [6,7,16,17], andnoncapsular polysaccha

rides [18,19] (see Chapter 1). Although it has beenreported [20] that lectin-binding may differentiate

between microbial genera, it is evident thatlectin-binding can be used as a

typing method to study intraspecies differences [8,21-32].Because lectins

Use of trade names is for identification only and does notconstitute endorsement by the

Public Health Service or by the U.S. Department of Healthand Human Services. 111 have the ability to bind to awide variety of microbial components that contain eithersimple or complex carbohydrates, they can be used todetect changes in the bacterial cell envelope, identifycell surface carbohydrates, or differentiate betweenstrains of bacteria on the basis of variations in cellsurface carbohydrates. The latter use has epidemiologicalimplications in that it can provide information that canbe used either directly or in conjunction with other

typing systems to study the population dynamics ofpathogenic microorganisms. This chapter will focus on theuse of lectins to study sexually transmittedmicroorganisms such as Neisseria gonorrhoeae, Haemophilusducreyi, Treponema pallidum subsp pallidum, and Treponemapallidum subsp pertenue. II. NEISSERIA CONORRHOEAE A. TheOrganism Neisseria gonorrhoeae is a gram-negativediplococcus that colonizes human mucosal surfaces.Gonococci cause symptomatic or asymptomatic localizedinfections including urethritis, cervicitis, proctitis,pharyngitis, and conjunctivitis. Disseminated infectionsoccur either by direct extension to adjacent organs[pelvic inflammatory disease (PID}, epididymitis], or bybacteremic spread (skin lesions, tenosynovitis, septicarthritis, endocarditis, and meningitis). B. CellEnvelope The outer membrane is composed of proteins,phospholipids, and lipopolysaccharide. Neisseriagonorrhoeae and other mucosal pathogens, such as N.meningitidis and Haemophilus inf/uenzae, produce highlybranched and relatively short lipopolysaccharides [33].These lipopolysaccharides lack the repeating and variable0-antigens that are characteristic of thelipopolysaccharide of the Enterobacteriaceae. To reflectthese structural differences, gonococcallipopolysaccharide and other bacteriallipopolysaccharidesthat share similar features are referred to aslipooligosaccharides (LOS). 1. Properties of GonococcalLipoo/igosaccharide Gonococcal LOS consists of severalcomponents with molecular weights ranging from 3200 to7100 [34]. Immunochemical analysis of gonococcal LOS usingmonoclonal antibodies has revealed the presence of discreteindividual LOS components [35,36]. Gonococcal LOS sharestructures and epitopes with human glycosphingolipids thatare precursors to blood group antigens [37]. These LOSare sialylated in vivo by host cytidinemonophospho-N-acetylneuraminic acid in a manner similar tohost glycosphingolipids

[38,39]. The sialic acid is readily lost during in vitrocultivation [40]. The

structures of the oligosaccharide component of the twomajor LOS compo

nents of N. gonorrhoeae strain F62 have recently beendetermined [41] and

are illustrated in Figure 1. These are the only LOSstructures that have

been completely determined. The terminal Gal,8-1,4GlcNAcm(see Fig. 1B)

is the structure that is sialylated in vivo [38]. Gonococciare apparently able

to vary the expression of LOS determinants [42]. The basisfor this varia

tion is incompletely understood.

C. Interaction with Lectins

The LOS is primarily responsible for the interaction of N.gonorrhoeae

with lectins, such as wheat germ agglutinin (WGA) [13].Schaefer et al. [26]

observed that WGA reacted with 165 of 165 strains of N.gonorrhoeae and

proposed that agglutination by WGA could be used as an aidin identifying

this organism. Yajko et al. [27] observed that rarestrains (4 of 126) were

not agglutinated by WGA and proposed that a combination ofWGA,

soybean agglutinin (SBA), and chromogenic substrates couldbe used for

the confirmatory identification of N. gonorrhoeae.Lectin-binding studies have been useful in determining thestructure of

the gonococcal LOS. Allen et al. [15] observed that somelectins [WGA,

SBA, Phaseolus vulgaris (PHA), and ricin] agglutinated allstrains, suggest

ing that ,B-linked o-N-acetylglucose-(GlcNAc) and,8-o-galactose (Gal) units

were common structural features of the LOS. Other lectins[Dolichos

bif/oris (DBA), limulin, and Sophora japonica] agglutinatedonly some

strains, suggesting that a-o-GalNAc units and ,B-o-Gallinked to GalNAc

GalNAc~l ~3Gal~l--+1G!cNAc~l ~4-Glc~l ~4Hepa~KDO 3 (A) iGlcNAcal ~2Hepal Gal~l~4GlcNAc~l~3Gal~l~4-Glc~l~4Hepa~KDO3 (B) i GlcNAcal ~ 2Hepal

Figure 1 Structure of the two major dioligosaccharidesderived from the lipooligo

saccharides of N. gonorrhoeae strain F62. (Data from Ref.41.) occurred as structural features of the LOS of somestrains. In retrospect, these results are consistent withthe recently determined structures shown in Figure 1.The differential binding of lectins toN. gonorrhoeae thatwas observed in previous studies [15,24] suggested thepresence or exposure of specific carbohydrates may varybetween strains, and these differences may be used toprovide useful epidemiological information. 1. MethodologyThe following protocol has been used successfully forlectin-typing of N. gonorrhoeae. Fourteen lectins withspecificities for the carbohydrates found in gonococcalLOS, including many of those used by Allen et al. [15] toobtain structural information about the gonococcal cellenvelope and by Doyle et al. [24] to distinguish betweenmembers of the family Neisseriaceae, were obtained ascommercially prepared lectin agglutination panels from EYLaboratories (San Mateo, California). The 14 lectins andtheir carbohydrate specificities are shown in Table 1.Gonococcal (GC) agar base (Difco Laboratories, Detroit,Michigan) containing 1 OJo (vollvol) IsoVitaTable 1Lectins Used in the Commercially Prepared Panels and theirCarbohydrate Specificities• Lectin Symbol SpecificitybConcanavalin A Con A a-D-Man > a-D-Glc > a-D-GlcNAc Lensculinaris LCA a-D-Man > a-D-Glc Trichosanthes kirilowiiTRK D-Gal Griffonia simplicifolia I GS-I a-D-Gal >a-D-GalNAc Arachis hypogeae PNA D-Gal-(3-(1-+3) >/3-D-GalNH 2 = a-D-Gal Glycine max SBA a-D-GalNAc >/3-D-GalNAc > a-D-Gal Dolichos bijlorus DBA a-D-GalNAc >a-D-Gal Griffoniil simplicifolia II GS-11 a-D-GlcNAc =/3-D-GlcNAc Solanum tuberosum STA (/3-D-GlcNAc)2-5 >(13-D-GlcNAc) Triticum vulgaris WGA (/3-D-GlcNAc) 3 >(13-D-GlcNAc) 2 > (13-D-GlcNAc) Limaxflavus LFAN-Acetylneuraminic acid Phaseolus vulgaris PHA D-GalNAcUlex europaeus I UEA-I a-L-Fuc Lotus tetragonolobus LOTUSa-L-Fuc "Lectin specificities according to Goldstein andHayes [7] and E-Y Laboratories. bMan, mannose; Ole,

glucose; Fuc, fucose; Gal, galactose. Source: Ref. 47.

Applications of Lectins to Agents of STD 115

leX enrichment (BBL, Cockeysville, Maryland) and 20Jo(vol!vol) fetal bo

vine serum (Hyclone Laboratories, Ogden, Utah), pH 7 .2,is the medium

of choice for cultivating gonococci for lectin typing. Theinclusion of

Hyclone fetal bovine serum had no effect on theagglutination patterns of

control strains of N. gonorrhoeae (unpublished data).Likewise, varying

the initial pH of the medium from 6.5 to 7.8 did notaffect the agglutination

patterns of control strains. Cultures of N. gonorrhoeaegrown on chocolate

agar for more than 18 hr often exhibited cell lysis andautoagglutination,

causing difficulty in interpreting lectin agglutinationpatterns [15]. This

can be prevented by using a commercially availableantiautoagglutination

reagent [27] or by incorporating deoxyribonuclease (DNase)in the suspend

ing buffer [15]. To reduce the effects of cell lysis andautoagglutination,

cultures of N. gonorrhoeae are incubated for 16-18 hr at35°C under aero

bic conditions with 5% C0 2 • For agglutination studies,cotton-tipped wood

applicator sticks are used to transfer cell growth totubes containing Tris

buffered saline (TBS), pH 7.5. For standardization, the

cell suspensions

are adjusted spectrophotometrically (535 nm) to theoptical density of a

McFarland No. 4 standard. This results in a suspensioncontaining about

10 9 colony-forming units (cfu)/ml, determined to beoptimum for providing

agglutination reactions that could be visually read withoutrequiring the

use of a magnifying device. A 50to 100-J.Ll volume ofcell suspension is added to each well in the

panel of dried lectins. The panels are placed on agyratory platform, rotated

at room temperature for 5-10 min, read for agglutinationactivity, and the

reactions recorded. Although each panel contains a cover toprevent drying

of the lectin-cell suspension, it is still very importantto use only the number

of panels that can be easily read and interpreted within a10to 15-min

period. Additionally, gonococcal strains with knownagglutination patterns

should be included as controls with each set of panel runsto determine any

lot-to-lot variation in manufactured lectin panels. Toensure the specificity

of the reactions, samples of carbohydrate solutionsspecific for each lectin

are added to the wells to demonstrate inhibition of theagglutination reac

tions. Also, gonococcal strains are blindly repeated ondifferent days to

ensure that agglutination patterns do not change over time.

2. Epidemiologic Applications

Vasquez and Berron [43] suggested that auxotyping [44] andserotyping

[45,46] were not an efficient means of discriminating amongmany gono

coccal isolates and proposed that adding the lectin-bindingpattern would

markedly increase the discriminating power. We have usedlectin agglutina

tion to study the characteristics of 150 strains of N.gonorrhoeae that were

isolated during epidemiological investigations inCalifornia, Hawaii, Georgia, and Pennsylvania [47].Twenty-four different agglutination patterns were observed(Table 2). All of the strains were agglutinated by lectins(TRK, SBA, and GS-I) that exhibit specificities for a-D-Galor a-DGalNAc. Conversely, none of the strains wasagglutinated by LFA, which has a specificity forN-acetylneuraminic acid (sialic acid). Interestingly, notall of the strains were agglutinated by lectins sharing aspecificity for D-galactose. For example, PNAagglutinated 149/150 isolates; whereas, DBA agglutinatedonly 57/150, and PHA agglutinated 7/150 isolates. It ispossible that the specific carbohydrate is either in thewrong conformation, is inaccessible to the lectin, or thatit is not present on every strain. Six isolates were notagglutinated with WGA; five of these strains were isolatedfrom patients with gonococcal meningitis and will bediscussed in detail later. The agglutination patternsobserved with the 150 isolates were designated as lectingroups. Table 3 shows that most strains (670Jo) belonged tolectin groups 6 and 7. These lectin groups accounted for70.6% of the isolates from California and Hawaii, 56.8%of the isolates from Georgia, and 72.7% of the isolatesfrom Pennsylvania. In this limited study, we were unableto observe any correlation between a specific lectin group,the sex of the patient, or geographic area. Fortunately,paired isolates were available for five couples fromGeorgia (Table 4). The auxotype and serovar agreed for allof the sexual partner isolates; however, the lectin groupagreed in only three of five isolate pairs. Among thedisparate pairs, the difference in lectin group

represented the loss and gain of reactivity to just asingle lectin. Interestingly, one male sexual partner(patient A) had four female partners, and the lectin groupwas the same in three of four female partners. In lightof the variability of gonococcal LOS, the role of hostfactors (e.g., antibodies) in LOS variation (and lectingroup variability) should be examined. Twelve pairs ofisolates from sexual partners were also available amongthe isolates from California and Hawaii (Table 5). Threeof four paired isolates belonging to different lectingroups also differed in either serovar or auxotype,suggesting that these may represent infections withmultiple strains. Approximately 15% of patients have beeninfected with more than one strain of N. gonorrhoeae[48]. The lectin group, serovar, and auxotype agreed for 5of 12 paired isolates; in only one pair were the auxotypeand serovar the same and the lectin group different.Previous studies have shown that the auxotype [49] andserovar [46] are relatively stable characteristics of aparticular strain of N. gonorrhoeae; therefore, it islikely that the recently described heterogeneity ofgonococcal LOS [42] is responsible for the variability oflectin agglutination patterns among partner isolates withidentical auxotypes and serovars. When the two prepon>Lectin 'E.. group Con A LCA TRK GS-1 PNA SBA DBA GS-IISTA WGA LFA PHA UEA-1 LOTUS ;::;~ s· 1 + + + + + ++ + :II "' 2 + + + + + + + Q 3 + + + + + ++ + + I'"" ID l"l 4 + + + + + + :r 5 + + + ++ + + "' 6 + + + + + + + Q > 7 + + + + + + ~ 8+ + + + + + + :II "' 9 + + + + + + + + Q 10 + + + ++ + + + Ill -1 11 + + + + + + + + + 0 12 + + + ++ + + 13 + + + + + + + + 14 + + + + + + + + + 15+ + + + + + + + 16 + + + + + + + + 17 + + + + + +18 + + + + + + + 19 + + + + + + + + + 20 + + ++ + 21 + + + + + + 22 + + + + + 23 + + + + 24 + ++ + + •con A, concanavalin A; LCA, Lens culinaris; TRK,Trichosanthes kirilowii; GS-I, Griffonia simplicifolia I;PNA, Arachis hypogeae; ... ... SBA, Glycine max; DBA,Dolichos biflorus; GS-11, Griffonia simplicifolia II; STA,Solanum tuberosum; WGA, Triticum vulgaris; LFA, ......Lim ax flavus; PHA, Phaseolus vulgaris; UEA-I, Ulexeuropaeus I; LOTUS, Lotus tetragonolobus. Source: Ref. 47.Table 3 Geographic Distribution of Lectin AgglutinationGroups No. of isolates from California Lectin group andHawaii Georgia Pennsylvania 1 0 1 0 2 0 2 0 3 0 0 42 0 5 0 1 0 6 13 12 9 7 23 13 31 8 1 0 9 0 010 0 0 11 0 0 12 1 1 9 13 3 4 0 14 0 1 0 15 1 1 016 0 1 0 17 1 0 0 18 0 1 0 19 1 0 0 20 1 0 0 21 00 4 22 1 0 1 23 2 0 0 24 0 derant lectin groupsrecorded for this study (Table 6) were examined, it was

observed that lectin group 6 and lectin group 7 could notbe differentiated based on lA or IB servovars. Although lAand IB serovars were found in both groups, IB serovarswere more frequently present in both groups. Auxotropicrequirements showed that most of these isolates requiredproline or were prototrophic. Plasmid analysis revealedthat all of the isolates contained the 2.6-MDa crypticplasmid, and most contained either no {3lactamase plasmid(3.2and 4.4-MDa plasmids), either a combination of the2.6-MDa cryptic plasmid, and the 4.4or 3.2-MDa {3-lactamaseplasmid, or the combination of the 4.4 {3-lactamaseplasmid and the 24.5-MDa

conjugative plasmid. No differentiation of these lectingroups could be

based on plasmid content. Lectins have also been used toretrospectively examine chromosomally

mediated penicillin-resistant (Pen') strains isolatedduring an epidemiologi

cal investigation in New Mexico [50]. Nineteen Pen'isolates of N. gonor

rhoeae were obtained from 8 heterosexual and 11 homosexualmen; 21

penicillin-susceptible (Pen') isolates were obtained from15 heterosexual

and 6 homosexual men. All of the patients resided in thesame area of

Albuquerque, New Mexico. Strains were characterized byserotype [45,46],

auxotype [44], plasmid content [51,52], and lectin group.The results are

presented in Table 7. All of the heterosexual isolatesbelonged to lectin

groups 7 and 12, with the exception of one isolate thatbelonged to lectin

group 25. Both the Pen' and Pen' heterosexual isolateswere distributed

between lectin groups 7 and 12. All of the Pen' and Pen'

isolates obtained

from homosexual men belonged to lectin group 7, however,indicating that

isolates infecting these homosexual men shared a commoncharacteristic.

Among these gonococcal isolates, eight were from homosexualpartners

and two from heterosexual partners (Table 8). All of thesexual partner

isolates belonged to lectin group 7, and all had 2.6-MDacryptic plasmids;

two isolates contained 24.5-MDa conjugative plasmids. Eightof the ten

sexual partners were the same serovar, whereas only fourof ten had similar

auxotrophic requirements. The two heterosexual partnersisolates that be

longed to lectin group 7 had the same serovar andauxotrophic require

ments. When all of the isolates from this study werecompared, the hetero

Table 4 Relationships Among Sexual Pairs, Serovars, Lectin

Groups, and Auxotypes for Georgia Isolates

Patient Sex• Serovar Lectin group Auxotype

A M IB-5 6 Prototrophic

B F IB-5 8 Prototrophic


c F IB-5 6 Prototrophic

D M IB-5 5 Prototrophic

E F IB-5 6 Prototrophic


E F IB-5 6 Prototrophic


F F IB-5 6 Prototrophic

"M, male; F, female.

Source: Ref. 47. TableS Relationships Among Sexual Pairs,Serovars, Lectin Groups, and Auxotypes for California andHawaii Isolates Patient No. Sex• Serovar Lectin groupAuxotype 1 M IB-5 6 Prototrophic 2 F IB-5 6Prototrophic 3 M IB-13 8 Prototrophic 4 F IB-5 7Prototrophic 5 F IB-1 6 Prob 6 M IB-1 6 Prob 7 FIA-3 22 Prototrophic 8 M IA-3 6 Prob 9 F IBC 6Prototrophic 10 M IBC 6 Prototrophic 11 F IB-5 18Prototrophic 12 M IB-5 6 Prototrophic 13 F IB-1 7Prototrophic 14 M IB-1 7 Prob 15 F IB-5 7 Prototrophic16 M IB-13 7 Prototrophic 17 M IB-5 7 Prototrophic 18 FIA-6 7 Prob 19 M IA-3 6 Prob 20 F IA-3 22Prototrophic 21 F IB-5 6 Prototrophic 22 M IB-5 6Prototrophic 23 F IA-4 7 Prototrophic 24 M IA-4 7Prototrophic •M, male; F, female. bPro, prolinerequiring. <unclassified in the present nomenclature forserological classsification. Source: Ref. 47. sexual Pen•isolates had proline growth requirements, whereas thehomosexual Pen• and Pen• isolates and the heterosexual Pen•isolates required either proline or were prototrophic.Homosexual and heterosexual Pen• isolates belonged toprotein IB serovars, whereas the homosexual andheteroxexual Pen• isolates belonged to protein lA and IBserovars. This was the first reported study in whichgonococcal isolates from the same geographic area showedthe same lectin agglutination group for the sexual partnersand that isolates obtained from different patientanatomical sites agreed with lectin group and serovar.

Table 6 Serovars, Auxotypes, and Plasmid ContentsAssociated with the Two Pre

ponderant Lectin Groups Plasmid content

Lectin group Serovar Auxotype• (MOab)

6(34t IA(l4) Arg/Pro (5), 2.6 (18) Pro (14) 2.6, 3.2 (1)IB(20) Arg (1) 2.6, 4.4 (5) Proto (14) 2.6, 4.4, 24.5(10)

7 (67) lA (25) Pro (24) 2.6 (42) Proto (34) 2.6, 3.2 (1)IB (42) Arg/Pro (6) 2.6, 4.4 (9) Ser (1) 2.6, 4.4, 24.5(15) Arg/Pro/Hyx (1) Pro/Met (1)

"Arg, arginine; Pro, proline; Ser, serine; Met, methionine;Hyx, hypoxanthine; Proto, proto

trohic.

bMDa, megadaltons.

~umber of isolates listed in parenthesis.

3. Lack of Wheat Germ Agglutinin Reactivity as a VirulenceMarker

Various studies have reported that between 3 and 50Jo ofgonococcal isolates

do not react with WGA [27,43]. The lack of reactivity withWGA has also

been noted among strains of N. gonorrhoeae associated withmeningitis, a

relatively rare complication of disseminated gonococcalinfection (DGI)

[53-57). In 1984, three cases of meningitis associatedwith disseminated

gonococcal infections were reported in Pennsylvania; oneof the patients

died of complications [58,59]. Neisseria gonorrhoeae wasisolated from the

Table 7 Lectin Agglutination Patterns of New MexicoPenicillin-Sensitive (Pen')

and Penicillin-Resistant (Pen') Gonococcal Isolates bySexual Preference

Lectin

group

7

12

25 Sexual preference Heterosexual Pen' 6 9 0 Pen' 43 1 Homosexual Pen' Pen' 6 0 0 11 0 0 Table 8Relationship of Lectin Group, Serovar, Plasmid Content, andAuxotype of Isolates for the New Mexico Sexual Partners•Sexual Culture partners Sexb site< Serovar Auxotype AM R IB-1 Prototrophic B M u IB-1 Proline c M u IB-1Proline D M R IB-1 Prototrophic E M u IB-4 PrototrophicF M R IB-4 Proline G M u IB-4 Prototrophic H M u IB-1Prototrophic I F c IB-1 Proline J M u IB-1 Proline"All isolates were in lectin group 7, and all had 2.6 MDaplasmids. bM, male; F, female. <R, rectum; U, urethra; C,cervix. Source: Data from Ref. 50. blood and cervix ofthe deceased patient, from the cerebrospinal fluid andcervix of the second patient, and from the blood of thethird patient. When examined with lectin panels, all fiveisolates failed to react with WGA. Fifty urogenital andtwo DGI arthritis-associated isolates of N. gonorrhoeaewere then obtained from the same Pennsylvania community andtested for agglutination by WGA; 49 of SO urogenital andthe 2 arthritisassociated isolates were agglutinated byWGA. Results of the lectin agglutination of the Smeningitis-associated, 50 urogenital, and 2 DOlarthritisassociated isolates obtained from this geographicarea, and 13 PID isolates obtained from the Centers forDisease Control and Prevention (CDC) culture collection areshown in Table 9. One isolate obtained from a case ofuncomplicated or urogenital gonorrhoea did not agglutinateWGA. The failure of WGA to agglutinate the strainsisolated from patients with gonococcal meningitis may be amarker for a virulence factor. A comparison of serovar andlectin reactivity with WGA between the S Pennsylvaniameningitis-associated isolates, 10 DOl isolates notassociated with meningitis and obtained from the CDCculture collection, and the SO Pennsylvania urogenitalisolates causing uncomplicated gonorrhoea is summarized inTable 10. The meningitis-associated isolates did notagglutinate WGA and were preponderantly lA serovars. TheDOl isolates not associated with meningitis were alsopreponderantly lA serovars; however, they were agglu


Table 9 Various Lectin Reactions with the PennsylvaniaN.gonorrhoeae Isolates

from Different Anatomical Sites No. of reactiveisolates/total DGI," DGI,

Lectin Symbol Urogenital PID" arthritis meningitis

Concanavalin A ConA 0/50 0/13 0/2 0/5

Arachis hypogeae PNA 50/50 13/13 2/2 515

Dolichos biflorus DBA 13/50 13/13 2/2 515

Solanum tuberosum STA 46/50 12/13 2/2 515

Triticum vulgaris WGA 49/50 12/13 2/2 0/5

"PID, pelvic inflammatory disease; DGI, disseminatedgonococcal infection.

Source: Data from Ref. 58.

tinated by WGA. The isolates from uncomplicated infectionswere prepon

derantly IB serovars, and only one isolate did notagglutinate WGA. Frasch

[13] observed that encapsulated strains of N. meningitidiswere not aggluti

nated by WGA, whereas nonencapsulated strains wereagglutinated by

WGA. No capsule could be demonstrated when the fivegonococcal isolates

from meningitis patients were examined further. Asubsequent study re

vealed that a gonococcal isolate associated withmeningitis, obtained from

a different geographic area of the United States, wasagglutinated by WGA

[60]. These findings suggested that the strains isolated inPennsylvania may

represent the spread of a single clone; however, this isunlikely, since sero

type analysis indicated that these strains belonged toboth lA and IB sero

vars.

Table 10 Comparison of Serogroup and Wheat GermAgglutination Among

Patient Isolates of N. gonorrhoeae

Diagnosis

DOl" /meningitis

DOl/without meningitis

Uncomplicated gonorrhoea No. of patients 5 10 50

"DOl, disseminated gonococcal infection.

Source: Data from Ref. 59. Protein I serogroup lA IB 41 8 2 6 44 Wheat germ agglutination yes no 5 1049 4. Use of Lectins to Assess Treatment Failure Anotherapplication of lectin typing has been in drug treatmenttrials to characterize preand posttreatment isolates ofN. gonorrhoeae. Two studies have been reported thatcharacterized preand posttreatment isolates followingenoxacin [61] or cefodizime versus cefotaxime [62] therapyfor gonorrhea. Lectin typing has been used in conjunctionwith auxotyping, serovar analysis, and minimum inhibitoryconcentration (MIC) to the investigational drug toascertain whether the preand posttreatment isolates wereidentical, thus representing either treatment failure,reinfection from an untreated partner, or emergence of anantibiotic-resistant strain. Nonidentical preandposttreatment isolates suggest reinfection with a differentstrain. Twelve pairs of preand posttreatment isolates wereavailable from the enoxacin trial. The results of thisstudy are shown in Table 11. Eleven of twelve pairs ofpreand posttreatment isolates had identical auxotypes andserovars; two isolate pairs differed in lectin group. Inone pair, the difference represented the loss ofreactivity with GS-11 and the gain of reactivity with DBA.In the other pair, the posttreatment isolate lostreactivity with GS-11. In addition, 11 of 12 pairs ofpreand posttreatment strains had the same MIC to enoxacin.One of the posttreatment strains exhibited a 67-foldincrease in the MIC value and probably represents theselection of a resistant strain. In the other studyinvolving treatment of uncomplicated gonorrhea in men andwomen with either cefodizime or cefotaxime [62], all ofthe preand posttreatment isolates were identical forauxotype, serovar, and lectin group. The results of thesestudies suggest that, with one exception, the

posttreatment isolates represent either treatment failureor reinfection from an untreated partner. Ill. HAEMOPHILUSDUCREYI A. The Organism Haemophi/us ducreyi is agram-negative, nonmotile rod that is the etiological agentof chancroid. Chancroid is one of the five classic venerealdiseases (gonorrhea, syphilis, lymphogranuloma venereum,donovanosis, and chancroid) and is characterized bypainful genital ulcers and frequent lymphadenopathy.Chancroid was considered to be a relatively uncommonsexually transmitted disease in the United States;however, the number of reported cases have increased morethan sixfold since 1984 [63]. Haemophilus ducreyi is amajor cause of genital ulcers in Asia and Africa, where theprevalence of chancroid often exceeds that of syphilis.This bacterium is difficult to isolate directly fromgenital lesions [63]. Sensitivity of culture is dependenton the medium, the specimen, and the Table 11 TypingPatterns for N. gonorrhoeae Strains Isolated Before andAfter Therapy with Enoxacin Pretreatment strainsPosttreatment strains Strain MIC Lectin MIC number(/Lg/ml) Auxotype" Serovar group (/Lg/ml) Auxotype"Serovar 1 0.03 NR IB-3 6 0.03 NR IB-3 2 0.06 ProlineIB-5 7 0.06 Proline IB-5 3 0.03 Proline IB-4 7 0.03Proline IB-4 4 0.06 Proline IB-4 7 0.06 Proline IB-45 0.06 NrPhe IB-6 7 0.06 NRPhe IB-6 6 0.03 ProlineIB-4 6 0.06 Proline IB-4 7 0.03 Prototrophic IB-3 60.03 Prototrophic IB-3 8 0.03 Prototrophic IB-2 72.00 Prototrophic IB-2 9 0.03 NRPhe IB-6 7 0.03NrPhe IB-6 10 0.06 Prototrophic NTb 12 0.06Prototrophic NT 11 0.03 NR IB-6 6 0.06 NR IB-6 12O.o3 NRPhe IB-6 12 0.03 NRPhe IB-6 "NR Phe, notrequiring and inhibited by phenylalanine; NR, norequirement. bNT, not typable. Source: Data from Ref. 60.Lectin group 6 7 7 7 7 6 6 7 7 6 6 7 > ,'2.. ;:r !!:. =· = "' Q .... tD !l :r "' Q >~ = "' Q Ill -1 c ... N 1;,11 expertise of thelaboratorian. Relatively little is known about theepidemiology of chancroid because there are relatively fewphenotypic characteristics that can be used todifferentiate among isolates of H. ducreyi. Outer membraneprotein profiles [64,65] and amino peptidase profiles[66,67] have been used, but do not provide suitablediscrimination. Recently, the taxonomy of H. ducreyi hasbeen called into question [68,69]. Sequence analysis ofribosomal RNA genes indicates that H. ducreyi belongs inthe family Pasteurel/aceae, but is not a member of thegenus Haemophilus [70]. It has been suggested that H.ducreyi may comprise a single-species genus and, thus, maybe of clonal origin. Therefore, it is not surprising thatit has been difficult to differentiate among strains of

H. ducreyi. The identification of restriction fragmentlength polymorphisms (RFLPs) among ribosomal RNA genes(ribotyping) has recently been useful in typing isolatesof H. ducreyi [71]. B. Cell Envelope In electronmicrographs, the outer membrane of H. ducreyi appearsmorphologically similar to those of other gram-negativebacteria [63,72]. The outer membrane profile is alsotypical of gram-negative bacteria, with one to severalproteins predominating [64,73,74]. Changes in growth mediumcomposition, atmospheric conditions, and temperature do notappear to affect the outer membrane protein profile;however, the electrophoretic pattern of the LPS isapparently affected by conditions of growth [75]. Thepresence of a capsule has not been adequatelydemonstrated. Bertram [76] observed the presence ofantibody-stabilized extracellular capsular material byelectron microscopy. In contrast, Johnson et al. [77]failed to observe extracellular acidic polysaccharide onthin sections of H. ducreyi stained with ruthenium red.1. Properties of Haemophilus ducreyi Lipoo/igosaccharidesHaemophilus sp. have been reported to produce both smoothand rough LPS [78]. Several investigators have recentlyexamined the type of LPS produced by H. ducreyi.Haemophilus ducreyi possesses a low molecular weight LPSthat contains heptose, ketodeoxyoctonate, glucose,galactose, glucosamine, and galactosamine [79]. It issimilar in size to that of other mucosal pathogens andtherefore, is referred to as LOS. The H. ducreyi LOS ismore toxic than the LOS from H. inf/uenzae or the LPS fromEscherichia coli, and has produced skin abscesses in animalmodels [80]. Two patterns of reactivity with monoclonalantibodies have been observed among isolates of H.ducreyi [81]. The LOS from 24 of 25 strains examined boundmonoclonal antibody 3Fll, which recognizes a terminalGal(j

1 ,4GlcNAc epitope that has a structure similar to humanblood group anti

gens [37,81]. This epitope is also present on somegonococcal LOSs and is

apparently sialylated in vivo [39]. Whether H. ducreyi LOSis sialylated in

vivo remains to be determined. One strain has beenidentified that does not

react with monoclonal 3Fll. This strain produces a smallerLOS than the

other strains that have been examined [81].

C. Interaction of Haemophilus ducreyi with Lectins

1. Methodology

Laboratory cultivation of H. ducreyi can be accomplishedwith heart infu

sion agar (Difco Laboratories) supplemented with 50Jorabbit erythrocytes,

10% fetal bovine sera (Hyclone Laboratories), and 1%IsoVitaleX (BBL).

Various media have been described for cultivating H.ducreyi [63,82-91].

After growth for 18 hr at 35°C in 5% C0 2 , cells weresuspended in TBS,

pH 7 .5, as described for N. gonorrhoeae. Unlike gonococci,H. ducreyi

cells form clumps or chains when in suspension thatinterfere in interpreting

lectin agglutination reactions. By allowing suspensions ofH. ducreyi to

stand for 1-2 min, cell clumps and chains of cells settleto the bottom of

the tube. The liquid at the top of the tube, containingsingle or pairs of

cells, is removed and transferred to another tube. Thissuspension can now

be adjusted spectrophotometrically (535 nm) to an opticaldensity of a

McFarland No. 4 standard. Samples (50J,tl) of the cellsuspension are added

to the lectin panel wells, panels are placed on a gyratoryplatform, rotated

for 5-10 min, and agglutination read. Additionally,

plastic panels contain

ing no lectins were used to place a suspension of eachisolate. With the aid

of a stereomicroscope (Bausch and Lomb) at 5 x or lOxmagnification,

the granulation between wells containing organisms andlectins could be

compared with the wells containing only organisms. In thoseinstances

when there was difficulty determining if agglutination wasoccurring, or

granulation was causing a false-positive reaction,specific carbohydrates

were used to show reversibility of agglutination.

2. Epidemiological Applications

A total of 63 isolates of H. ducreyi obtained fromoutbreaks in California,

Florida, Georgia, New York, and Massachusetts wereexamined. The re

sults of the H. ducreyi lectin agglutination activity areshown in Table 12.0f

the 63 isolates examined, two preponderant lectin groupswere observed.

Differences in lectin agglutination focused on thereactivity of isolates with

GS-11. Among these isolates, 27 (43%) did not agglutinatewith GS-11,

whereas 36 (57%) isolates did agglutinate with this lectin.What appeared

to be agglutination activity with LCA and PHA could not bereversed Table 12 Lectin Agglutination Patterns of 63Haemophilus ducreyi Isolates Geographic Number Lectin"location and of year of isolation Isolates ConA LCA TRKGS-I PNA SBA DBA GS-11 STA WGA LFA PHA UEA-I LOTUS

California Orange County (1982) 5 + + + + + + + +8 + + + + + + + + + Long Beach (1987) 5 + + + + ++ + + + Georgia Atlanta (1958) 1 + + + + + + + + + 1+ + + + + + + + Atlanta (1981) 3 + + + + + + + + + 3+ + + + + + + + New York New York City (1985) 6 + ++ + + + + + 7 + + + + + + + + + ... 1-.J = Cll n::r !.. iii Ill :I Cl. ~ Q ... ; Boston (1985) 4+ + + + Florida West Palm Beach (1983) 7 + + + + ++ + + Orlando (1985) 1 + + + + + + + + Jacksonville(1986) 4 + + + + + + + + Ohio Cleveland (1985) 2 ++ + + Texas Dallas (1987) 3 + + + + •Lectinabbreviations shown in Table 1. + + + + + + + + + + + ++ + + + + + + + + + + + + + + + + + + + + + + ++ + + + + + > "5!.. ;::;~ o· :I II> Q I""'I'D 1"1 :r II> Q > (JQ I'D :I II> Q VI ~ c.... 1-.1 loCI with specific carbohydrates. It wasconcluded that this activity was due to nonspecificclumping that resulted in false-positive agglutination. Theresults presented in Table 12 are different from thosepresented by Korting et al. [92]. In that study, all ofthe isolates were agglutinated by LCA and PHA, with someagglutination activity reported for DBA, UEA-1, and Lotus.In contrast, we were unable to show agglutination activityby LCA, DBA, PHA, UEA-1 or Lotus. However, all of theisolates were agglutinated by TRK, GS-1, PNA, SBA, andSTA. Similar to the results reported by Korting et al.[92], some isolates were agglutinated by GS-11. Nodifference was found in agglutination activity of isolatesrelative to geographic location in the United States. Thesimilarity in lectin agglutination patterns for theseisolates may suggest that these outbreaks were caused by afew strains; however, the inability of lectins todifferentiate many agglutination types among the H.ducreyi strains examined is consistent with data suggestingthat few phenotypic properties exist that can be used forstrain typing [63]. IV. TREPONEMA PALL/DUM SUBSPECIESPALL/DUM AND TREPONEMA PALL/DUM SUBSPECIES PERTENUE A.The Organism Treponema pallidum subsp. pallidum (T.pallidum) and T. pallidum subsp. pertenue (T. pertenue)are nonculturable (pathogenic) spirochetes that are thecausal agents of syphilis and yaws, respectively. Theybelong to the family Spirochaetaceae, genus Treponema.Syphilis is a chronic disease that occurs throughout theworld. Infection within the host is characterized by aprimary lesion or soft chancre that usually develops withina few weeks. Treponemes can often be observed by directexamination of lesion fluid by darkfield microscopy.Treponema pertenue is the causative agent of yaws,transmission occurs through skin contact, and the diseaseoccurs primarily in the tropics. Both T. pallidum and T.

pertenue are gram-negative organism and are considered tobe microaerophilic [93,94]. Treponema pallidum takes upoxygen and possesses an electron transport system [95,96].Both microorganisms exhibit corkscrew motility that isaccompanied by rotation around the longitudinal axis and abending or flexing. The ends of these spirochetes aresomewhat pointed or tapered [97 ,98]. Electron microscopyhas shown that these treponemes possess an outer membrane,also referred to as an outer envelope, which covers theaxial filaments located on the surface [97,98].Antigenically, these treponemes are considered identical,and a species-specific antigen has not yet been identified.Pathogenic and nonpathogenic treponemes can bedifferentiated by DNA homology, but


this method does not differentiate between T. pallidum andT. pertenue

[99,100].

B. The Outer Envelope and Cell Wall

The pathogenic treponemes are nonculturable, althoughlimited success has

been obtained with tissue cell cultures [101-103]. Becauseit is difficult to

grow pathogenic treponemes in vitro, very littleinformation is available

concerning their biochemical reactions, metabolism, andchemical composi

tion, although the lipid composition of T. pallidum wasreported by Ma

thews et al. [104]. Some reports have indicated that thepathogenic trepo

nemes possess a surface acidic polysaccharide [105-107],but other reports

question whether this polysaccharide is synthesized by thetreponeme, or is

derived from host tissue [1 08-111]. The polysaccharidepossesses glucose

and galactose residues [ 104, 107, 11 0].

C. Interaction with Lectins

Very little information is available about the interactionof T. pallidum or

T. pertenue with lectins. Baseman et al. [112] used lectinsnoncovalently

bound to Leighton tube coverslips to bind freshly extractedtreponemes

and facilitate the removal of cellular debris and fluids.They observed that

T. pallidum bound to lectin film coverslips containingConA, WGA, PHA,

and pokeweed mitogen (PWM). Binding ofT. pallidum to ConAoccurred

with greater affinity than to WGA and PHA. Fitzgerald andJohnson [109]

reported that T. pallidum interacted with WGA and SBA, andthey postu

lated that the ligand was the acidic polysaccharide. Thesurface polysaccha

ride of T. pallidum is reported to be composed ofhyaluronic acid and

chondroitin sulfate or related acidic glycosaminoglycans[107]. Hyaluronic

acid, consisting of N-acetyl-o-glucosamine-o-glucuronicacid, will interact

with WGA. Similarly, chondroitin sulfate consists ofN-acetyl-o-galac

tosamine-o-glucuronic acid, which will interact with SBA.These acidic

polysaccharides are present in syphilitic lesions[108-111], and it is unclear

whether the polysaccharide material is part of thetreponemal cell surface,

or results from host tissue damage during the infectionprocess [108-111].

1. Methodology

Since T. pallidum and T. pertenue cannot be easilysubcultured in vitro,

growth of the organisms is accomplished by intratesticularpassage in

healthy rabbits. Rabbit testes are excised 11-14 dayspostinfection. The

treponemes are harvested by slicing the testes, placingthem in a solution of

0.02 M phosphate-buffered saline (pH 7 .2), containing0.075 M sodium

citrate, and teasing the treponemes out of the testiculartissue with gentle agitation. The pathogenic treponemesare separated from other cellular debris by densitygradient centrifugation in 200Jo Percoll (Pharmacia FineChemicals, Piscataway, New Jersey). Treponemes purified bydensity gradient centrifugation are washed three times withTBS, pH 7.5, to remove contaminating Percoll. Treponemaphagedenis biotype Reiter was cultivated in NIHthioglycolate broth (Difco Laboratories) containing 10%inactivated normal rabbit sera. Growth of thisnonpathogenic treponeme was accomplished at 35°C for 4-7days. Purified pathogenic treponemes and the nonpathogenicT. phagedenis were suspended in TBS, pH 7.5, adjustedspectrophotometrically to yield a suspension equivalent toa McFarland No. 4 standard, and examined for agglutinationactivity with the panel of 14 lectins. Normal rabbittesticular extract (NRTE) was prepared by pulverizingnitrogen-frozen testicular tissue in a stainless steelmill. The powdered testicular tissue was extracted threetimes with TBS, pH 7.5, and layered onto 20% Percoll fordensity gradient centrifugation as described earlier. TheNRTE and 20% Percoll were also examined for agglutinationactivity with the 14 panellectins. 2. EpidemiologicalApplications Three strains of T. pallidum and two strainsof T. pertenue, all isolated from different geographicareas, and one nonpathogenic strain were examined forlectin reactivity. The results of lectin agglutination

with the treponemal strains are shown in Table 13. Thesame lectin agglutination pattern was observed for all T.pallidum and T. pertenue strains. The pathogenictreponemes reacted with ConA, GS-1, SBA, and WGA. The NRTEalso showed reactivity with these same lectins. It isuncertain whether the treponemal reactivity with theselectins is due to treponemal cell surface carbohydrate,or to contaminating testicular tissue present on thesurface of the treponemes. The pathogenic strains couldnot be differentiated from the T. phagedenis biotypeReiter owing to the uncertainty of testicular tissuecontamination. Agglutination of lectins with thepathogenic treponemal suspensions was reversed withspecific carbohydrates, indicating specific reactivity;however, with the uncertainty of testicular tissuecontamination, the specificity of these reactions mayreside in contaminating testicular material. The 200JoPercoll preparation did not show lectin agglutination. V.CONCLUSIONS The information reported in this chapterrepresents only preliminary information concerning theapplications of lectins to some organisms associated withsexually transmitted diseases. All agglutinationcharacteristics of these microorganisms were determinedusing prepared panels of 14lectins. Fur.... l.ol l.olTable 13 Lectin" Agglutination Activity of PathogenicTreponema! Strains, Compared with One NonpathogenicTreponema! Strain Con A LCA TRK OS-I PNA SBA DBA GS-11STA WGA LFA PHA UEA-1 LOTUS T. phagedenis + + + + + + +biotype Reiter T. pa/lidum subsp. pa/lidum Strain 1 + ++ + + + + + + ± + Strain 2 + + + + + + + + + ± +Strain 3 + + + + + + + + + ± + T.pallidum subsp.pertenue Strain 1 + + + + + + + + + ± + Strain 2 + ++ + + + + + + ± + 200Jo Percoll NRTEb + + + + + + ++ "See Table 1 for identification of lectins. ~RTE,normal rabbit testicular extract . ther characterizationof cell surface carbohydrates and their role in cellsurface structure may require the use of more-specificlectin reagents than those contained in panels used forstudies reported in this chapter. The agglutinationreactions of N. gonorrhoeae, based on structuralcomponents of the LOS, were carbohydrate-specific andcould be inhibited. The agglutination of N. gonorrhoeaeappeared to be due to the availability of exposed terminalstructures of the carbohydrate residues. Masking of somecarbohydrate structures or influence of a neighboringstructural charge phenomenon may offer an explanation whylectins of similar specificity do not agglutinate the samegonococcal strains. Twenty-four different lectin groupswere observed in these studies. It has been suggested thatgonococcal LOS epitopes may undergo phase variation, which

may, in turn, affect lectin agglutinability [42].However, subculture did not appear to affect lectinagglutination of N. gonorrhoeae strains used in thesestudies. Strains of N. gonorrhoeae were repeatedlysubcultured and used as blind controls. The originalagglutination pattern of each strain did not change aftersubculture. The observation that differences in lectinagglutination with GS-11 and DBA may be suggestive ofstructural differences in a-o-GalNAc or a-o-GlcNAcresidues, lectin accessibility to residues, absence ofthese residues in some strains, or the population ofresidues presented to lectin reagents. In the studiespresented in this chapter, two strains of N. gonorrhoeaeagglutinated with ConA, which represented an uncommonlectin agglutination pattern among the 150 strainsexamined. In contrast, Vazquez and Berron [43] observed amore frequent agglutination of N. gonorrhoeae strains byConA. Perhaps these strains, which were isolated indifferent geographic areas of the world, exhibited moreLOS structural differences than the 150 strains examinedfrom the United States. The strains isolated in theUnited States showed infrequent agglutination with UEA-1,LCA, and PHA, when compared with the agglutination patternsreported by Vazquez and Berron [43]. The data presentedin this chapter concerning lectin agglutination differencesamong strains of N. gonorrhoeae represent a differenttyping system that can be used in conjunction withserotyping and auxotyping. The use of lectinagglutination, serotyping, and auxotyping can be useful inclinical studies to determine antibiotic treatmentfailures, emergence of antibiotic-resistant strains, andreinfection from an untreated sexual partner. Strains ofH. ducreyi reported in this chapter did not show a highdegree of variability in lectin agglutination patterns.Repeated subculture of these H. ducreyi strains did notaffect the reproducibility of lectin agglutinationpatterns, suggesting that subculturing did not affect LOSepitopes present on the cell surface. That only twolectin agglutination patterns for

the isolates of H. ducreyi were available for examinationin this study is

consistent with results of studies indicating fewphenotypic characteristics

are available for differentiating strains of thisorganism. Studies of lectin

agglutination with H. ducreyi reported by Korting et al.

[92] with strains

isolated from other world geographic areas did showdifferences in lectin

agglutination. At least 20 different lectin agglutinationpatterns were re

corded, suggesting that there may be methodologicaldifferences or differ

ences in phenotypic characteristics among these strains.The lectin agglutination patterns of the pathogenictreponemes were

identical. No differences in lectin agglutination wereobserved between the

strains examined. A previous report indicated that lectinagglutination with

T. pallidum was due to acidic polysaccharides [107].However, whether this

polysaccharide is of treponema! origin or host tissueorigin is still unclear

[1 08-111]. Difficulty in obtaining high yields oftreponemes for lectin ag

glutination or in preparing pathogenic treponemes to ensureabsence of

host tissue fluid or debris will be a continual problem toproduce reliable

and accurate lectin agglutination results. Otherpreliminary studies have been initiated to examine otheragents

of sexually transmitted diseases. With the same panel of14lectins, strains

of Chlamydia trachomatis have been examined foragglutination by lectins.

Strains of C. trachomatis were agglutinated by lectinsspecific for a-o-Gle,

a-D-Gal, and a-o-GalNAc. McCoy tissue culture cells used

for passage of

C. trachomatis were also agglutinated by the same lectins,however, raising

concern that the agglutination may be due to contaminationby McCoy

tissue culture cell epitopes. Additionally, the panel of14 lectins has been

used to determine lectin agglutination patterns ofMycoplasma hominis.

Only after proteolytic treatment were M. hominis strainsagglutinated by

lectins specific for a-o-Gle, a-n-Gal, and a-o-GalNAc, inaccord with re

sults reported by Kahane and Tully [113]. Schiefer et al.[114], however,

did not observe agglutination of M. hominis strains bylectins, and treating

of these strains with pronase failed to enhanceagglutination reactivity.

Further examination of these microorganisms is necessaryto determine the

suitability of lectin applications and to determine thestability and repro

ducibility of lectin agglutination activity. Theapplication of lectin agglutination to agents of sexuallytransmit

ted diseases may facilitate the detection of interandintrastrain variations.

ACKNOWLEDGMENTS

The authors thank R. E. George, E. F. Hunter, and S. A.Larsen for their

technical assistance and guidance in growth of pathogenictreponemes, and

21. Cole HB, Ezzell JW Jr, Keller KF, Doyle RJ.Differentiation of Bacillus anthracis and other Bacillusspecies by lectins. J Clin Microbiol 1984; 19:4853.

22. Curtis GDW, Slack MPE. Wheat-germ agglutination inNeisseria gonorrhoeae. Br J Vener Dis 1981; 57:253-255.

23. · Davidson SK, Keller KF, Doyle RJ. Differentiation ofcoagulase-positive and coagulase-negative staphylococci bylectins and plant agglutinins. J Clin Microbiol1982;15:547-553.

24. Doyle RJ, Nedjat-Haiem F, Keller KF, Frach CE.Diagnostic value of interactions between members of thefamily Neisseriaceae and lectins. J Clin Microbiol1984;19:383-387.

25. Senne JE. Lectin agglutination of Neisseriagonorrhoeae. Clin Microbiol Newsl1981; 3:10.

26. Schafer RL, Keller KF, Doyle RJ. Lectins in diagnosticmicrobiology: use of wheat germ agglutinin for laboratoryidentification of Neisseria gonorrhoeae. J ClinMicrobiol1979; 10:669-672.

27. Yajko DM, ChuA, Hadley KW. Rapid confirmatoryidentification of Neisseria gonorrhoeae with lectins andchromogenic substrates. J Clin Microbiol 1984; 19:380-382.

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97. Hovind-Hougen K. Determination by means of electronmicroscopy of morphological criteria of value forclassification of some spirochetes, in particulartreponemes. Acta Pathol Microbiol Scand Suppl1976;255:1-41. 98. Fitzgerald TJ, Cleveland P, Johnson RC,Miller JN, Sykes JA. Scanning electron microscopy ofTreponema pallidum (Nichols strain) attached to culturedmammalian cells. J Bacteriol1977; 130:1333-1344. 99. MiaoRM, FieldsteelAH. Genetics of Treponema: relationshipbetween Treponema pa/lidum and five cultivable treponemes.J Bacteriol1978; 133: 101-107. 100. Miao RM, FieldsteelAH. Genetic relationship between Treponema pallidum andTreponema pertenue, two noncultivable human pathogens. JBacteriol 1980; 141:427-429. 101. Fieldsteel AH, Cox DL,Moeckli RA. Cultivation of virulent Treponema pallidum intissue culture. Infect lmmun 1981; 32:908-915. 102.Fieldsteel AH, Cox DL, Moeckli RA. Further studies onreplication of virulent Treponema pa/lidum in tissuecultures of SflEp cells. Infect Immun 1982; 35:449-455.103. Norris SJ. In vitro cultivation of Treponema pa/lidum:independent confirmation. Infect Immun 1982; 36:437-439.104. Mathews HM, Yang TK, Jenkin HM. Unique lipidcomposition of Treponemapallidum (Nichols virulent strain).Infect Immun 1979; 24:713-719. 105. Fitzgerald TJ, JohnsonRC, Wolfe ET. Mucopolysaccharide material resulting fromthe interaction of Treponema pa/lidum (Nichols strain) withcultured mammalian cells. Infect Immun 1978; 22:575-584.106. VanDerSluis JJ, Ten Kate FJ, Vuzevski VD, Stolz E.Light and electron microscopy of rabbit testes infectedwith Treponema pallidum (Nichols strain): nature ofdeposited mucopolysaccharides and localisation of

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4

Application of Lectins in

Clinical Bacteriology

MALCOLM SLIFKIN Allegheny General Hospital and MedicalCollege of

Pennsylvania, Pittsburgh, Pennsylvania

I. INTRODUCTION

The application of lectins for the identification ofvarious microorganisms

was first brought to the attention of the microbiologistthrough the investi

gations of Sumner and Howell, in 1936 [1]. Theseinvestigators observed

that various lectins capable of agglutinating agglutinatederythrocytes could

be inhibited by simple sugars [2]. These observations werethe first to pres

ent evidence that the lectin from the jackbean,concanavalin A (ConA),

agglutinated certain species of Mycobacterium andActinomyces. The lipids

of Mycobacterium paratuberculosis, when extracted withacetone and ether

and incorporated in a salt suspension, were reported toagglutinate in the

presence of ConA. This latter report of Sumner and Howell,therefore,

may be viewed as one that brought forth the initialmomentum of lectin

application into the realm of clinical microbiology aswell as into many

other facets of microbiology. Bacterial lectins have beendemonstrated to be associated with a role

in infectious disease [3]. The bacterial surface lectinsserve as molecules of

recognition in cell-cell interactions [2,4]. The reader isthus referred to

interactions [5]. There are many reviews on lectins andapplications of these unique

proteins [6-17]. At present, there are several reviews onthe application of

lectins as diagnostic reagents for use in clinicalmicrobiology [18-23]. This

chapter will (1) review the pertinent applications ofnonbacteriallectins for

the identification or classification of variousgram-positive and gram143 negative bacteria and (2)present examples of the use of lectins as diagnosticprobes. Special attention is devoted to the presentationof methods applicable to the detection of variousbacteria, by lectin interaction, for the clinicalbacteriology laboratory. II. USE OF LECTINS FORGRAM-POSITIVE BACTERIA A. Bacillus Concanavalin A reacts

with a wall polymer of B. subtilis, represented bypolyglucosylglycerol phosphate teichoic acid [24]. Variouslectins have made it possible to eliminate the relativelyexpensive and time-consuming culturing or serologicaltesting. For example, the laboratory identification of B.anthracis has traditionally been a time-consuming andinexact exercise for several reasons. These problemsinclude overlap of common antigens, an overlap for 'Yphage receptor sites, and the need for special media andreagents. Some lectins bind to plastic surfaces allowing aprocedure to be developed known as an enzyme-linkedlectinosorbent assay (ELLA) (see Chapter 1). This assaywas demonstrated to detect as few as 26,000 cells of B.anthracis employing horseradish peroxidase-conjugatedsoybean lectin [25]. Soybean, Abrus precatorius andGriffonia simplicifolia (GSA-1) agglutinins were reportedto agglutinate B. anthracis and B. mycoides [25].Bacillus anthracis will bind with fluorescein-conjugatedsoybean agglutinin [27]; however, B. mycoides does notdivide at 37°C, so a Bacillus culture incubated at 37°Cand agglutinated by soybean agglutinin may be considered tobe B. anthracis [18]. Confirmation of B. anthracis dependson the determination of exotoxins or on the cellwall-associated polysaccharide [27]. A procedure using asnail lectin, Helix pomatia, and the soybean lectin forthe differentiation of B. anthracis can be found in thefourth edition of the Manual of Clinical Microbiology,published in 1985 [27]. This agglutination procedure,reported by Cole et al. [26], can be performed withminimal reagents and can be completed in only minutes. ABacillus isolate mixed in a solution of soybean lectinwill agglutinate within 1-2 min if the bacterium is B.anthracis or B. mycoides. A second lectin, Helix pomatia,is positive for only B. mycoides. Lectins have also beenused to classify many strains of the insect pathogen B.thuringiensis [28]. B. Staphylococcus and Related BacteriaA relatively wide spectrum of gram-positive bacteria havebeen examined for their respective affinity for plantoranimal-derived lectins. Various lectins have proved usefulreagents for probing structural features of poly145

saccharides. Lectins can recognize N-acetyl-o-galactosamine(GalNAc)

structures. These lectins appear to respond to differencesof terminal aor

/3-galactose or aor /3-GalNAc [29]. Some a-GalNAc-specificlectins are

Dolichos biflorus, Griffonia simplicifolia, and Salviasclarea. The lectins

derived from Helix pomatia, Vicia villosa, Wistariajloribunda, and Gly

cine max react with aas well as /3-linked GalNAc residues.Concanavalin A was reported to precipitate witha-glucosylated, but

not with {3-glucosylated teichoic acids, from Staph.epidermidis by agar

diffusion [30]. This lectin reacted with only a-linkedN-acetylglucosamine

teichoic acids from strains of Staph. aureus. ConcanavalinA is reported to

precipitate the teichoic acid associated with a strain ofStaph. aureus that

contains a-N-acetyl-o-glucosaminyl residues, but does notprecipitate with

teichoic acids of other strains containing/3-N-acetyl-o-glucosaminyl groups

[31]. Staphylococcus aureus and the related bacteriumMicrococcus lyso

deikticus were reported to react with concanavalin A [32).Staphylococcus

aureus was reported to be agglutinated by a fraction ofhorseshoe crab

distinct from limulin [33]. Purified lectin from thehorseshoe crab Limulus

polyphemus agglutinates Staph. aureus cells [33].Generally, most laboratories employ the catalase andcoagulase tests,

along with Gram stain reaction, for the differentiation ofStaph. aureus

from the coagulase-negative staphylococci [34]. Coagulaseproduction is

reported to be associated with false-negative [35) andfalse-positive re

sponses [35,36]. A battery of lectins have beendemonstrated to be useful

for the differentiation of coagulase-negative andcoagulase-positive staphy

lococci [37]. This method provides a means todifferentiate these two

groups of staphylococci in only 5 mins. Staphylococcusaureus will not

agglutinate in the presence of the lectin from cells ofthe horseshoe crab

L. polyphemus nor the combination of lectins derived fromthe mango,

Mangifera indica, and from wheat germ, Triticum vulgaris.Agglutination

with these lectins occurs with the coagulase-negativestaphylococcal strains

tested. This slide agglutination test correctlydifferentiated 29 strains of

Staph. aureus and 30 strains of coagulase-negativestaphylococci.

C. Streptococcus

Most strains of Strep. mutans tested were reported to beagglutinated by

concanavalin A [38-40]. Enhancement of this agglutinationresponse was

achieved by addition of sucrose or addition of dextranaseto the bacterial

cells grown in the presence of sucrose. Inhibition studiesindicated that

different binding sites on the bacterial cells fromvarious serogroups were

responsible for the binding of this lectin. Otherinvestigations have reported

similar conclusions [39). In another investigation, ConAwas reported to bind to isolated cell walls of variousstrains of Strep. mutans [41-42]. Streptococcus mutansgrown in sucrose medium bound more ConA than those grown inglucose medium. After treatment with dextranase, thesucrose-grown cells bound twoto fourfold more of thelectin [42]. The albumen gland of the edible snail, H.pomatia (HPA), contains an agglutinin that specificallybinds to group A human erythrocytes, with a specificitydirected to terminal nonreducing N-acetyl-o-galactosamineresidues [43-46]. The lectins derived from H. pomatia andthe plants, Dolichos bif/orus (DBA) and Wisteriafloribunda (WF A), will specifically agglutinate group Cstreptococci owing to their high affinity forN-acetylgalactosamine, the major group-specificpolysaccharide of group C streptococci. There are manyother lectins that are effective in distinguishing group Cstreptococci from the other clinically significantiS-hemolytic streptococci (Table 1). In earlyinvestigations, crude extracts containing H. pomatialectins were shown to identify group C streptococcispecifically, without agglutination responses from eithergroups A, B, C, F, or G streptococci. The lectins from D.bif/orus and W. floribunda also agglutinate group CiS-hemolytic streptococci, without reactivity with otherclinically important serogroups [47]. Prokop et al. [48]determined that group C streptococci were specificallyagglutinated by the lectin from the H. pomatia. Theactivity of this lectin is directed toward terminal aand!3-N-acetyl-o-galactosamine residues. Group C streptococciwere reported to be agglutinated by the lectin from thealbumen gland of the snail Cepaea hortensis [49] and fromthe seeds of the tropical leguminous plant D. bif/orus[44,46]. One report has demonstrated, on the basis of morethan 4000 strains of iS-hemolytic streptococci, that H.pomatia was highly specific in agglutination of group Cstreptococci [48]. More recently, an investigation thatcomprised 1045 strains of group C streptococci, includingStrep. equisimilis, Strep. zooepidemicus, and Strep.dysgalactiae, were agglutinated by the Helix lectin [48].None of 12,264 strains of group A, 1346 strains of groupG, 330 strains of group B, as well as other streptococcalgroups were agglutinated. A few strains of Strep.anginosus (S. millen) associated with group G-specificcarbohydrate, agglutinated in the presence of the Helixlectin. Streptococcus sanguis strains with group Hantigens were also agglutinated with this lectin [ 48].

The H. pomatia lectin can react, however, with otherbacteria in addition to group C streptococci. The lectinhas been reported to agglutinate all strains ofCorynebacterium diphtheriae, some but not all Bacillus sp.,Salmonella, Escherichia coli 025, 088, 0117, 0126, and moststrains of Staph. aureus [46,48]. Table 1 Reactivity of13-Hemolytic Streptococci with Various Lectins LectinLycopersicon esculentum Solanum tuberosum Daturastramonium Momordica charantia Helix pomatia Dolichosbif/orus Wistaria floribunda Sambucus nigra Tulipasp:Limax flavus Common name Tomato Potato JimsonweedBitter pear melon Edible snail Horse gram WistariaElder bark Tulip Slug Carbohydrate specificity[inhibitor(s)] 13-1,4-linked GlcNAc containing three tofour saccharide units 13-1,4-linked oligomers of GalNAc13-1 ,4-linked oligomers of GalNAc Gal> GalNAc a-D-GalNAca-D-GalNAc, D-Gal N-acetyl-D-galactosamine Sialic acida-2,6Gal/GalNAC, lactose Complex glycoproteins Sialicacid Reactivity with groups A B c F + + -+ + + +± ± + + + + G .... :r Ill :r Q :r ;::;·!!!... = ~ 1"1 .... tD ... cs· 0 (IQ -< ... ""''I 148 The fishes Salmo irideus, Lucioperca lucioperca,and Rutilus rutilus contain a rhamnose-specific lectinthat agglutinates strains of streptococcal group G,variant strains of groups A and C, some strains ofstreptococcal groups D, E, L, 0, S, and T, as well assome salmonellae of the groups B, C, D, and E [48].Another lectin of R. rutilus is specific for o-glucosylresidues and agglutinates streptococci of group E,carrying 13-o-gluocosides and rhamnose. The lectin ofCyprinus carpio will agglutinate another subgroup ofgroupE streptococci and is specific foro-glucose [48]. Incontrast to the lectin from H. pomatia (HPA), theselectins do not agglutinate all of the strains mentionedand cannot be applied to routine diagnosis [48]. They canbe used only for the determination of immunochemicalend-grouping. The seed extracts of W. floribunda alsospecifically agglutinate group C streptococci, as shown bythe investigations of Ottensooser and associates [47].These authors emphasize the advantages of lectins inrelation to the savings of time, such as pretreatment ofbacteria for immunization, injection of bacteria intoanimals, and' absorptions of antisera to eliminatenonspecific antibody. Our first introduction with lectinsconcerned the development of a test that used threefluorogenic 4-methylumbelliferyl substrates and the lectinfrom D. biflorus (DBA). The assay provided a rapid,simple, and specific means to identify groups A, B, C, F,and G streptococcal isolates from throat cultures [50].The three methylumbelliferyl substrates used in that

investigation provided a means to accurately identifygroups A, B, and F 13-hemolytic streptococci isolates fromthroat cultures. Some strains of group C and Gstreptococci, however, could not be differentiated becauseof overlapping 4-methylumbelliferyl substrate patterns. Theuse of soluble preparation of the lectin DBA as anagglutination reagent provided a rapid and specificagglutination response when mixed on a microscope slidewith five or more colonies of group C organisms. Resultswere obtained within 30 sec of a hand rotation on amicroscope slide. No agglutination was detected with groupG or with groups A, B, or F streptococcal cells. The useof the combination of the methylumbelliferyl substrates andthe lectin reagent provided a nonserological method thatpermitted accurate identification of isolated colonies of13-hemolytic streptococci obtained from throat cultures.The methylumbelliferyl-lectin protocol was later applied byother investigators for the identification of streptococciisolated from cows with mastitis [51]. As with theprevious investigations [50], the use of the lectin D.biflorus was required to identify and differentiate Strep.dysgalactiae (group C) from the other groupablestreptococci. The demonstration of species-specific enzymeactivities of the streptococci with various4-methylumbelliferyl-conjugated substrates in combinationwith the lectin agglutination

Lectins in Clinical Bacteriology 149

permitted differentiation within 1-2 hr at 37°C. This is incontrast with the

standard identification techniques of these serogroupswith biochemical

tests that require an incubation period of 4-24 hr. Theseinvestigations led to the development of tests employinglectins

as diagnostic tools for the identification of variousserogroups of the (3

hemolytic streptococci. Thus, it was later reported thatcrude extracts of

DBA, when coupled to polystyrene particles with a spacearm, yielded

an effective lectin-latex microsphere reagent thatspecifically agglutinated

group C streptococcal antigens prepared as nitrous acid,autoclaved, or

enzyme extracts. The coated beads could also agglutinatesuspensions of

isolated colonies [52]. This latex-lectin preparation couldbe used to replace

latex microspheres conjugated to antibody to group Cstreptococci for use

in test kits for the identification of (3-hemolyticstreptococci from isolated

colonies. Many lectins are extremely stable at ambienttemperatures. The lectin

of W. floribunda was absorbed onto polystyrene latexmicrospheres that

were stained blue. Fifty microliters of these lectinconjugated microspheres

were placed on cardboard slides and dried overnight atambient tempera

ture. The dried lectin reagent cards were mixed with 50ILl of phosphate

buffer (pH 7 .2) at various times and tested with nitrousacid extracts of

group C streptococci. Strong and highly specificagglutination responses

were observed with the polystyrene lectin reagent dried oncards 7 months

previously. No reactions were observed with group Astreptococci or with

other clinically significant (3-hemolytic streptococci(Fig. 1). The use of HP A was used as a membraneparticle-capture assay to

detect nitrous acid-extracted group C streptococci. TheHPA lectin-latex

microsphere reagent was spotted onto a porous membrane anddried.

Group C streptococcal nitrous acid extract was added ontoa lectin-capture

substrate and incubated for 5 min at room temperature.Peroxidase labeled

HP A was then added to sandwich the analyte, and colordetection proceeded,

as with an enzyme-linked immunosorbent assay (ELISA) format(Fig. 2). Holm and associates have shown that blocking thesialic acid of the

type-specific polysaccharide of a type III strain of groupB streptococcal

cells by the sialic acid-specific lectin from the snailCepaea hortensis, leads

to the promotion of phagocytosis of a group Bstreptococcus of type Ia

strain [53]. The effect of the lectin was dose-dependentand required the

presence of complement. The sialic acid-specific lectinfrom C. hortensis

and C. nemoralis agglutinates all group B streptococcalstrains that contain

a type-specific polysaccharide [49,54]. However, group Bstreptococci that

lack a type-specific polysaccharide, as well as strains ofother streptococcal

groups and many other bacteria, including speciescontaining sialic acid,

are not agglutinated [53]. 150 Slifkin Figure 1Reactivity of dried polystyrene latex microspheresconjugated to Wisteria floribunda for a nitrous acidextract of group C streptococci. The microspheres havebeen dried on a cardboard card. The microspheres on thebottom left of the card have been mixed with a nitrousacid extract of group A streptococci and show no

agglutination, whereas the microspheres on the bottomright have agglutinated in the presence of group Cstreptococcal extract. A relatively wide spectrum oflectins were demonstrated to agglutinate various serotypesof group B streptococci [55] . In some instances treatmentof the bacterial cells with sialidase removed thereactivity of various lectins with those cells. In otherinstances, agglutination with lectins occurred only afterenzyme treatment of the bacterial cells. Group Bstreptococci can be specifically agglutinated or labeledwith fluorescein-conjugated lectins (Fig. 3) derived fromcertain plants of the Solanaceae family [56]. The pulplectin from the tomato, Lycopersicon esculentum (LYE),as well as the related plant, the potato, Solanum tuberosum(ST A), can be passively coupled to amide-modifiedpolystyrene microspheres [56]. These reagents could be usedfor the specific identification of group B streptococcigrown in selective Todd-Hewitt broth, such as Lim's broth,as well as nonselective broth [57]. These lectins do notagglutinate the other clinically significant serogroupablestreptococci. The lectins could 151

possibly be of value for their ability to assist in theidentification of group

B streptococcal colonization in women and infants. Thesetwo lectins have

an affinity, in part, for N-acetylglucosamine, the majorgroup-specific

polysaccharide for group B streptococci. Either the lectinof the tomato (LYE) or potato (ST A) conjugated to

latex particles will agglutinate group B streptococci.Particulate organisms,

as well as nitrous acid, autoclaved, or enzyme extractswere agglutinated or

precipitated, respectively, by STA. These streptococci,like those of group

A and group C streptococci, contain relatively high amountsof N-acetyl-o

glucosamine, compared with members of groups D, F, or G[58]. The

unique presence of 1 ,4-anhydroglucitol in the cells of

group B streptococci

[59] may, in part, be related to the affinity of the tomatoand the potato

lectins for group B streptococci. It has been shown thattomato lectin binds

oligosaccharides containing the repeating disaccharide({j-1 ,4-GlcNAc-{j),

or poly-N-acetyllactosamine sequence, requiring at leastthree repeating

disaccharide units for interaction [60]. Although notprecisely defined, the relevant antigenic determinants of

group B polysaccharide appear to be associated with therhamnose-glucitol

Figure 2 Microsphere-enzymatic procedure using H. pomatialectin as a capture

reagent for a nitrous acid extract of group Cstreptococci. The microparticle-capture

membrane on the left did not indicate a positive reactionwhen group A nitrous

acid extract was added to the membrane. A positiveenzymatic response is seen on

the membrane on the right when group C nitrous acid extractwas added. figure 3 Fluorescent response of group Bstreptococci labeled with fluoresceinconjugated L.escu/entum. units or terminal rhamnose on side chainsassociated with N-acetylglucosamine [59]. It is possiblethat the tomato and potato lectin can interact with othersubstrates associated with group B streptococci. The lectinfrom the garden snail Cepaca hortensis has a specificityfor sialic acid-containing polysaccharides and can reactwith group B streptococci [53]. Various typeable andnontypeable strains of group B streptococci were examinedto determine their respective reactivity to LYE coupled topolystyrene spheres (Table 2). All the strains grown ineither Todd-Hewitt or Lim's broth reacted with the lectinreagent. Nitrous acid, Streptomyces a/bus lysozymeenzyme, and autoclaved extracts of group B streptococcalcells yielded results similar to those with particulategroup B streptococcal cells grown in these culture broths.

None of the type 1b strains grown on Columbia sheep bloodagar, however, reacted with the LYE-conjugatedmicrospheres. Observation of medium-dependentagglutination reactions have been previously reported withLegionella-lectin interactions [61]. The latterinvestigators [61] emphasized that when lectins areemployed as diagnostic reagents, growth conditions must becarefully standardized. Fluorescein-conjugated LYE labeledall the group B streptococcal cells grown from the bloodagar medium. The serotype 1 b strains, however, did notfluoresce as brightly as did the other group B serotypes.No fluorescence or agglutination response were observedwhen the other serogroups of 153

streptococci were examined. The lectin from the solanaceousplant Datura

stramonium did not agglutinate group B streptococci northe other sera

groups tested. Datura stramonium, similar to LYE and STA,is a member of the

Solanaceae family of plants. This lectin, like that of thetomato and potato

lectins, is specific for A, B, and 0 erythrocytes andbinds to chitooligosac

charides [60]. The lack of group B streptococcal activitywith D. stramo

nium may be related to the inability of relatively smallconcentrations of

N-acetyl-o-glucosamine to block the agglutination of humanerythrocytes

with this lectin, or to its lack of reactivity with dimersor trimers of N

acetyl-o-glucosamine [60]. It was previously reported thatvarious commercially available serogro

uping reagents cross-reacted with the Todd-Hewitt brothmanufactured by

Difco Laboratories, but not with that manufactured byBaltimore Biologi

cal Laboratories [62]. The tomato or potato lectins do notprecipitate in

inoculated Todd-Hewitt broth of either manufacturer.Three elderberry species contain two identicalcarbohydrate-binding

sites per molecule and exhibit a very high specificity forthe Neu5Aca-2,6

Gal/GalNAc sequence [63-65]. Relative affinities forvarious oligosaccha

rides were significantly different among them, suggestingdifferences in the

detailed structure of the carbohydrate-binding sites ofthese related lectins.

Previous to these reports, no plant lectin had beenreported to bind specifi

Table 2 Agglutination of Group B Streptococci withLycopersicon esculen

tum or Solanum tuberosum Lectin Coupled to PolystyreneSpheres Agglutination-response (no. agglutinated/no.tested) No. Columbia sheep Todd-Hewitt Lim's

Organisms tested blood agar broth broth

Nontypeable 2 0/2 2/2 2/2

Ia 4 3/4 4/4 4/4

lb 4 0/4 4/4 4/4

Ic (la/c) 4 4/4 4/4 4/4

II 4 4/4 4/4 4/4

III 4 4/4 4/4 4/4

Group A 28 0/28 0/28 0/28

Group C 20 0/20 0/20 0/20

Group F 20 0/20 0/20 0/20

Group G 20 0/20 0/20 0/20 cally to sialic acid or to

oligosaccharide units that included this carbohydrateresidue [66]. Sialic acids represent about 30 derivativesof N-acetylor N-glycolylneuraminic acids [67]. Varioussialic acid derivatives exhibit interesting speciesandtissue-specific distribution. Sialic acids play animportant role as receptors for viruses [68] as well asin the social behavior of cells. They also act as maskingagents on antigens, receptors, and other recognition sitesof the cell surface [16]. There is a relatively smallselection of lectins that specifically bind to sialicacids [67]. The lectin isolated from elderberry, Sambucusnigra (SNA), binds the sequence NeuNAca-2,6-o-Gal/o-GalGalNAc with high specificity [63]. Thislectin has a relatively weak affinity for o-galactose oro-GalNAc, but does not bind to either NeuNAc or NeuNGuc.It has a high affinity for terminal sialic acid that islinked a~2,6to o-galactose, compared with the a-2-3-linkedisomer [63]. On the basis of inhibitions with simplesugars, the elderberry bark lectin was demonstrated to bespecific foro-galactose [63]. The lectin from the tulip(Tulipa; TUG) is derived from the bulb of the plant, amember of the family Liliaceae [69]. This lectin wasreported to be the first lectin to be isolated from thisplant family and is the first example of aphytohemagglutinin obtained from a monocotyledonous speciesother than the Graminaeae family associated with wheat,rye, barley, and rice [69]. Two lectins with differentagglutinating affinities are associated with T. gesnerianabulbs [70]. One TUG lectin component will agglutinatetrypsinized human or rabbit erythrocytes [69], but willnot agglutinate nontreated human red blood cells [69]. Asecond TUG lectin was previously shown to agglutinate thecells of the Saccharomyces genus at a concentration of15-30 ~-tllml, but did not agglutinate various species ofCandida, E. coli, Staph. aureus or B. subtilis [71]. Thelack of agglutination response of group A streptococcalextracts with TUG-bound latex particles appears toindicate that the lectin substrates may be either denaturedor not extractable. The group A streptococcal extracts, incontrast, agglutinated latex particles bound with antibodythat is directed to the specific group antigen or"C-substance" reported to be N-acetylglucosamine [58]. Inthe literature, the tulip lectin has been reported not tohave an affinity for this glycoprotein [69,71]. Accordingto one investigation, o-mannose, o-mannose-6-phosphate,L-fucose, and L-fucosylamine were demonstrated to be potentinhibitors of this lectin in the binding to Saccharomycescerevisiae cells [70]. In a hapten-inhibitioninvestigation, agglutination of human erythrocytes withTUG lectin was inhibited by N-acetylgalactosamine, lactose,

fucose, and galactose [69]. 155 It has been determinedthat the TUG lectin derived from the bulb, or

the lectin from the bark of Sambucus nigra, the elderberry,will agglutinate

both group A and C streptococci (see Table 1).Agglutination of group A

streptococcal organisms was affected with tulip lectin at aconcentration of

0.3 mg/ml on latex spheres. All of the 40 stains of group Astreptococci

grown on Columbia sheep blood agar agglutinated with thislectin reagent

in 15-30 sec by manually rocking the reactants on a glassmicroscope slide.

Within this period, the reactions appeared at anagglutination reactivity

strength of 2-3 + (Fig. 4). The tulip lectin reagentagglutinated 12 of the 40 (300Jo) test strains of

the group C streptococci. These relatively weakeragglutination responses,

however, occurred after 30-60 sec of manual rocking of thereactants. The

reactions of these group C strains produced only 1 +agglutination re

sponses within this observation period. All 40 strains ofgroup A strepto

cocci were also agglutinated in the presence ofmicrospheres prepared with

0.01 mg of lectin per milliliter. These 2-3 + agglutinationreactions were

observed within 30-60 sec of manual rocking of thereactants on micro

scope slides. Furthermore, at this lower concentration oftulip lectin, none

Figure 4 Agglutination of group A streptococci with Tulipalectin conjugated on

microspheres. The lectin-conjugated particles on the leftwere mixed with group C

streptococci. The right side shows the lectin-microphaseand group A streptococci. 156 of the group Cstreptococcal strains agglutinated within 60 sec ofobservation. Conjugation of the TUG lectin onto 0.2-J.'mpolystrene microspheres also yields agglutination withgroup A streptococci and none with the other clinicallysignificant serogroups. In contrast, the use of largermicrospheres effected no differentiation between group Aor C streptococci (Table 3). None of the strains of groupB, F, or G streptococci agglutinated in the presence ofthe TUG-latex reagent. The use of different lots ofColumbia sheep blood agar yielded the same agglutinationpatterns with these streptococci. Nitrous acid, enzyme,autoclave, or sonic-derived extracts of the various13-hemolytic streptococcal serogroups did not produceresponses when tested with the TUG-latex reagent. Theseextracts, however, did produce a specific agglutinationresponse when mixed with their respective commerciallyprepared serodiagnostic-latex reagent. Inhibition studieswere performed to determine the effects of various sugarsand glycoprotein inhibitors on the reactivity of TUGlectin, with either group A or group C streptococci. Thisinhibition protocol was performed by preparing twofoldserial dilutions of various sugars and glycoproteins inphosphate buffer, as shown in Table 4. These preparationswere respectively added to 0.1 ml of TUG-latex reagent.The latter contained 30 1-'g of lectin per milliliter. Thelectin and inhibitors were mixed on a glass microscopeslide with a wooden applicator stick for 10 sec. Four tosix colonies of group A or group C streptococci wereremoved from a sheep blood agar plate with a woodenapplicator stick and mixed into the lectinlatex inhibitormixture for 30 sec. Four to six colonies of group Astreptococci were also mixed into 0.1 ml of TUG-latexreagent that did not contain any potential inhibitorsolution to serve as a control. A determination of Table3 Effect of Microsphere Size on Specificity and ReactionTime with Interaction of Tulip Lectin and Group A andGroup C Streptococci Reaction time (sec) Latex particleSerogroupA Serogroup C size {J£m) 30 60 90 30 60 90 0.73+" 3+ 3+ 1+ 1+ 2+ 0.5 2+ 3+ 3+ 1+ 1+ 2+ 0.3 2+ 2+ 3+ 1+0.2 1+ 2+ 3+ "Strength of reaction: -, no agglutination;1 + , many fine clumps; 2 + , a few moderate-sized clumps;

3 +, many moderate-sized clumps. 157

Table 4 Inhibitory Effect of VariousCarbohydrate-Glycoproteins on the Aggluti

nation of Tulip Lectin to Groups A and C Streptococci

Carbohydrate/ glycoprotein

Asialofetuin

Fetuin

N-Acetylmuramic acid

N-Acetylneuraminic acid

N-Acetyl-o-galactosamine

Methyl-{j-o-galactopyranoside

Lactose

Mannose

Fucose

Group C streptococcal nitrous acid extract Minimalconcentration required for inhibition of tulip-mediatedagglutination of group A streptococci (mM)" 0.2 0.53.0 1.5 NI NI NI NI NI NI

"NI, no inhibition at concentrations less than 200 mM.

agglutination was made after manually rocking the slide for1 min. The

inhibition studies demonstrated the various sialoproteinsare effective as

inhibitors of agglutination by TUG-microsphere reagent,with either group

A or group C streptococci (see Table 4). In contrast,various simple sugars

or oligosaccharides do not inhibit this agglutinationresponse. Groups A

and C streptococci have been reported to contain slightly

higher concentra

tions of the sialoprotein and N-acetylmuramic acid thangroups B, D, F, or

G streptococci [58]. Furthermore, the lectin ofLimaxflavus, which is very

specific for sialic acid [72], will also agglutinate onlygroups A and C

streptococci (see Table 1). Accordingly, there appears tobe no interaction

of TUG lectin for either N-acetylglucosamine orN-acetylsialolactosamine

(see Table 4). Thus, group A streptococcal binding sitesassociated with tulip lectin

appear not to be entirely related to the polysaccharidesestablished as the

respective group-specific antigens [58]. Therefore, onemay consider that

these binding sites may be associated, in part, with thesegroup-specific

polysaccharides as well as other substrates. All lectinmolecules are multiva

lent and have two or more carbohydrate binding sites[8,17]. However,

binding of lectins cannot always be fully correlated withtheir reported

biochemical specificities [6,13,74]. An extensivedetermination of all poten

tial inhibitors may not always be possible. Lectin affinitymay also be partly

related to the method used to purify the lectin [7]. 158A similar association of binding of groups A and Cstreptococci was reported with wheat germ agglutinin (WGA)[75]. This lectin has an affinity forN-acetyl-o-glucosamine and its derivatives [76]. The wheatgerm lectin was demonstrated to agglutinate all strains of

groups A and C streptococci tested [75]. Some strains ofgroups B, E, H, L, 0, and S streptococci also agglutinatein the presence of this lectin. As with the tulip andSambucus nigra lectins, the binding of groups A and Cstreptococci may not depend on a terminal position ofGlcNAc [75]. Primary colonies of group A streptococci canbe accurately identified with the use of tulip lectin asa lectin-bound latex reagent. The tulip lectinreagent thuscan be applied as an alternative method for the rapididentification of group A streptococcal colonies. Thelectin provides a relatively cost-effective means toidentify colonies of group A streptococcal isolates. Theuse of the tulip lectin, similar to the previouslyreported D. bif/orus lectin [52], affords the availabilityof a diagnostic reagent that can be prepared withrelatively less complexity than the production of anantibody for serogrouping procedures. Table 1 shows asummary of some of investigations on the interactions ofvarious lectins with serogroups A, B, C, F, and G{j-hemolytic streptococci. Kohler and Nagai [48] havereported that some {j-hemolytic strains of Strep.anginosus (Strep. millen) can react with HPA. They observedthat not all strains reacting with group-specific anti-Cantiserum were agglutinated with HPA. It was suggestedthat N-acetyl-o-galactosamine is not in the terminalposition. The agglutination by the HP A by nongroup CStrep. milleri strains also indicated that other antigensmay be involved. All eight strains of group C-associatedStrep. anginosus strains examined reacted with H. pomatiabound to polystyrene spheres (Table 5). This lectinreagent did not agglutinate group A, group F, ornongroupable Strep. anginosus strains. These reactionswere similar when either nitrous acid extracts ornonextracted cells were used. The TUG lectin reacted withall eight strains of group A Strep. anginosus and 250Joof the group C streptoTable 5 Interaction of VariousLectins with Streptococcus anginosus Lectin TulipaSambucus nigra Helix pomatia A 8/8 6/8 0/8 Serogroupof S. anginosus (no. tested/no. reactive) c FNongroupable 2/8 7/8 8/8 0/6 0/6 0/6 0/4 0/4 0/4

coccal strains. No reactivity was observed with the group For nongroup

able strains. The SNA-latex reagent reacted with most ofthe group A and

C strains of Strep. anginosus tested. This reactivity fornon-Strep. angino

sus group C streptococci on the SNA lectin was notpreviously observed

[48], which suggests the presence of other SNA-bindingsubstrates on the

Strep. anginosus strains. The lectin from Misgurnusanguillicaudatus has a relatively strong af

finity for rhamnose [77]. This lectin, therefore, may be ofvalue in the

identification of streptococci [22].

D. Listeria

Recently, Sonnenfeld and Doyle [see 18] reported that therelatively com

mon serotypes of L. monocytogenes could be differentiatedwith the use of

a panel of lectins. Four lectins, including the applicationof a lectinlike seed

extract (Persea americana), were demonstrated to be usefulin distinguish

ing the major serotypes of L. monocytogenes. The lectinsincluded in the

panel were Griffonia simplicifolia, Vicia villosa, HPA,and ConA. The

agglutination procedure required that the L. monocytogenesisolates be

heated to expose the lecting-binding sites of theorganisms.

Ill. USE OF LECTINS FOR GRAM-NEGATIVE BACTERIA

A. Enterobacteriaceae

Concanavalin A agglutinates a variety of gram-negativebacteria. The bind

ing site of this lectin appears to be related to thecarbohydrate moieties on

the lipopolysaccharide molecule [74]. Polysaccharides ofvarious serotypes

of Salmonella precipitate with ConA [78], and it can beapplied in micro

scopic slide agglutination tests [79]. This lectin has beenreported to react

with Salmonella strains containing the 0 : 1 factor, andit will also bind to

E. coli [80]. This lectin, therefore, could be employed toclassify lipopoly

saccharides derived from Salmonella, although notalllipopolysaccharides

presumed to contain a-o-glucosyl side chains form aprecipitate with ConA.

No precipitate was observed when ConA was reacted with thelipopolysac

charide from Sal. typhi [81]. The polysaccharide isreported to contain side

chains similar to Sal. typhimurium [78] with which faintprecipitation was

detected [78]. Concanavalin A was reported to precipitatelipopolysaccharide ex

tracts obtained from Shigella flexneri and Sal.abortivoequina, but not Sal.

enteritidis [81]. Yersinia entercolitica strains,possessing the antigenic factor

0: 12 are agglutinated with ConA [79]. In the genusErwinia, the strains of 160 Slifkin E. amylovora willagglutinate in the presence of ConA in contrast with E.caratovora and E. chrysanthemi [79]. Concanavalin Aconjugated with fluorescein did not react with Pseudomonasaeruginosa, Haemophilus influenzae, or Proteus mirabilisstrains [82]. The application of lectins for theidentification of bacteria commonly associated with ocularinfections has been demonstrated. Some lectinbindingpatterns were reported to be specific for the bacterialpathogens examined [82]. Litchi chinensis lectin was

demonstrated to be applicable for the identification ofmany 13-hemolytic strains of E. coli and of Proteus sp.isolates [83]. The H. pomatia lectin failed toprecipitate the lipopolysaccharide derived from Sal.typhimurium rough mutants of type Ra and Rb, but wassomewhat reactive with an SR mutant [84]. The H. pomatialectin [48] and the lectin from the horseshoe crab Limuluspolyphemus [85] also have been reported to agglutinateE. coli and other members of the Enterobacteriaceae.Salmonella minnesota cells were agglutinated with wholeLimulus serum, but did not agglutinate in the presence inL. polyphemus serum purified by affinity column procedure[86]. The sialic acid-binding lectin from the horseshoecrab Carcinoscorpius rotunda cauda, also known ascarcinoscorpin, will agglutinate E. coli K12 and Sal.minnesota R595 cells [86]. Vibrio cholerae does notagglutinate in the presence of this lectin. B.Pseudomonas A lectin obtained from the potato was reportedto distinguish strains of P. solanacearum [87]. Strainsof this bacterial species that are pathogenic for thepotato do not react with this lectin, whereas avirulentstrains of this species are agglutinated. Theinvestigators determined that an extracellularpolysaccharide, associated with virulent strains, appearsto inhibit the reactivity of the lectin with its ligandsite on a lipopolysaccharide. Sixteen serotypes of P.aeruginosa were typed with a panel of lectins [88]. Allserogroups were highly agglutinated by the lectin ofMoringa olifera seeds, except serotype H7. Differentiationof the various serotypes was accomplished withagglutination (and precipitation) tests when a panel oflectins derived from M. olifera, Artocarpus integrifolia,and Artocarpus lakoocha and lipopolysaccharide extractsof various serotypes of P. aeruginosa were used. Previousinvestigations on 9 strains of P. cepacia with arelatively large panel of lectins indicated aheterogeneity of lectin-binding sites [89]. All 161

strains did not react with the lima bean lectin. The latterresearch demon

strated that this lectin interacted only with P. cepacia,suggesting that this

lectin may be species-specific. The investigatorsrecommended that more

pseudomonad isolates be examined to confirm thisobservation.

C. Legione/la

A panel of plant lectins gave differential reactivitieswith many of six sera

groups of L. pneumophila tested [61]. The agglutinationpatterns appeared

not to be related to the serogroup of these bacteria, as itwas determined

that five of the strains of serotype 1 had uniqueagglutination patterns.

These investigations suggested that the cell surfaces ofLegione/laceae do

not contain carbohydrate groups that are accessible forbinding by lectins.

Some of these bacteria were agglutinated by lectinlikesubstances from

Persea americana, Mangijera indica, Aloe arborescens, andAlbizzia juli

brissin. These plant extracts are associated withpolyphenol derivatives,

tannins, and may be capable of binding cell surfaces [90].

D. Neisseria and Related Bacteria

Among the gram-negative bacteria, the application oflectins as diagnostic

reagents is especially associated with the members of thegenus Neisseria.

The use of wheat germ lectin to agglutinate strains of N.gonorrhoeae has

been reported [91-92]. Nonencapsulated N. meningitidis andN. gonor

rhoeae will also agglutinate in the presence of thislectin, whereas encapsu

lated N. meningitidis will not [93]. This observationsuggested that gono

coccal agglutination with wheat germ lectin may be relatedto the lack of

capsular material on the gonococci. Other investigationsalso have shown

that wheat germ lectin is of use for the identification ofN. gonorrhoeae

[94]. From an panel of 14 lectins, all strains of thegonococcal isolates

examined were agglutinated by wheat germ, soybean, andricin lectins [95].

None of the differential patterns of gonococciagglutination by lectins was

related to GC serogroups. The authors concluded that GCserogroup anti

genic determinants involve portions of sugar structurethat are larger than,

or separate from, those recognized by lectin-combiningsites. It was sug

gested that agglutination by these lectins is mediated bycell envelope lipo

polysaccharides, and this interaction involved elements ofcommon lipo

polysaccharide core structures. A panel of 22 lectins wasused to demon

strate the diagnostic effect of lectin on theidentification of the family

Neisseriaceae [96]. DeHormaeche et al. [97] determinedthat the lipopolysaccharide of

N. gonorrhoeae, but not nonpathogenic neisseriae, containedan epitope

consisting of N-acetylglucosamine andN-acetylgalactosamine, and was re162 active with wheatgerm agglutinin. Other investigators, using a battery oflectins, have suggested that the lipopolysaccharides from

pyocin-sensitive and pyocin-resistant strains of N.gonorrhoeae possesses terminal GlcNAc or Gal(or GalNAc)residues [95,98]. The use of wheat germ agglutinin forthe identification of N. gonorrhoeae was associated withnonspecific agglutination [94] and autoagglutination ofgonococcal suspensions [96]. Doyle et al. [96] employedwheat germ and soybean agglutinins in conjunction with aa-glutamylaminopeptidase assay. All of the gonococcalstrains and lectin-reactive meningococci weredistinguishable by the hydrolysis ofa-glutamyl-13-naphythylamide by the former. This assay isapplicable for distinguishing Moraxella cat arrha/is, N.lactamica, and N. meningitidis. The autoagglutinationphenomenon was overcome by DNase treatment of thebacterial cells. Other investigators used both wheat germand soybean agglutinins in conjunction with variouschromogenic substrates [99]. They identified N.gonorrhoeae and differentiated it from other Neisseria sp.examined. Recently, an investigation of 140 strains of N.gonorrhoeae has shown that 4.90Jo of the strains are notagglutinated with WGA [100] as previously reported [99].The use of ten lectins, however, yielded discriminatinggroups of serotypes lA and lB and prototroph andproline-dependent auxotypes [100]. This method of typingwas reproducible and may be considered for epidemiologicalapplications. E. Campy/obacter (Helicobacter) In a recentinvestigation, it was determined that C. fetus strainslacking surface array proteins with type Alipopolysaccharide were agglutinated with Griffoniasimplicifolia II, H. pomatia, and wheat germ lectins. Noagglutination was observed with C. fetus mutants thatlacked surface array proteins with type B or type Clipopolysaccharide or strains containing a surface arrayprotein layer [101]. The authors concluded that knowledgeof the inhibition of lectin-binding to lipopolysaccharideby the surface array proteins will aid in thecharacterization of the lipopolysaccharide interaction. Inanother investigation, various serotypes of C. jejuni andC. coli displayed differential reactivities withgalactose-binding lectins, the reactions of which appearedto be strain-specific [102]. Other investigators have usedlectins for the differentiation of various Campylobactersp. Strains of C. jejuni and C. coli interact specificallywith certain lectins and blood group antibodies [103].Agglutination by lectins was also reported effective forthe differentiation of C. jejuni and C. coli [104]. Asimple and effective agglutination procedure using a panelof lectins was developed for differentiating strains ofvarious species of 163

Campylobacter [104]. All strains tested were agglutinatedby the protein

reactive agglutinins of Mangifera indica (mango) andPersea americana

(avocado). A large number of the Campylobacter strains alsoagglutinated

in the presence of ConA and Triticum vulgaris. Thereactions of the other

lectins used in the investigation varied. The data impliedthat lectins could

be used in a supplementary procedure for fingerprintingindividual isolates.

IV. USE OF LECTINS FOR MYCOBACTERIA

Fluorescein-conjugated ConA was effective in thevisualization of Myco

bacterium fortuitum and M. chelonei in both pure cultureand experimental

keratitis samples from corneal scrapings [105]. Thislectin yields only mini

mal background staining of corneal tissue [106].Concanavalin A binds to

nonreducing terminal o-arabinofuranosyl residues associatedwith the end

chain of arabinogalactan of the cell wall of M. bovis [107].

V. EPIDEMIOLOGICAL APPLICATIONS

Applications of lectins for epidemiological investigationsand strain charac

terization of Haemophilus ducreyi [108], Campy/obacterjejuni [103], and

N. gonorrhoeae [109,110] have been reported. Theseinvestigations have

been applied in certain instances for clinical studies to

differentiate between

treatment failure and reinfection; to study transmission,and to investigate

the presence of subtypes of various pathogens. In-depthcoverage of this

topic is found in Chapter 3.

VI. CONCLUSIONS

The selection of a lectin as a potential diagnostic reagentfor use in bacteri

ology first requires that a reasonable protocol befollowed. Thus, the selec

tion of a lectin or a panel of lectins may be based on theaffinity of these

lectins for a particular marker for a given bacterialspecies. The perfect

example is clearly seen with group C streptococci and thereactivity of

those lectins for N-acetylgalactosamine. This relation oflectin with group C

streptococci yields a very effective means to discriminateone serogroup

from other clinically significant serogroups of~-hemolytic streptococci.

This group of streptococci contains relatively largeramounts of GalNAc in

comparison with that of other serogroups. The reactivityof the lectins that

bind with this carbohydrate moiety is not specific,however, for only this

one serogroup. These lectins can also bind to othergram-positive and gram

negative bacteria. Thus, the observation of characteristicgram-positive

morphology, catalase reactivity, and hemolytic responseprovides the 164 primary guidelines that establishes theuse of these lectins for ?-hemo? lytic streptococci.Without these first-stage guidelines, a lectin, such asthat of Helix pomatia, that differentiates group Cstreptococci from other ~-hemolytic streptococci can alsointeract, for example, with Staph. aureus [31]. On theother hand, the determination that LFA and TUG arerelatively effective for their respective binding withgroup B and group A streptococci is not based on thereactivity of these lectins for the antigenic determinantsgenerally associated with the serogroups. Instead,reactivity of these lectins for the serogroups does notappear to be for the N-acetylglucosamine determinants,but possibly for sialic acid residues as well as othersubstrates. Furthermore, chemical modification of thelectin, the lectin concentration, as well as the testassay system are some of the factors that will influencethe potential for a lectin to be used as a diagnosticprobe. Relative to microsphere size, it was recentlyreported that the use of relatively small-diameterpolystyrene particles will decrease the sensitivity of alatex agglutination test [111]. For tulip lectin, however,small microspheres yield a highly specific response togroup A streptococcal organisms. This, in part, may be dueto possible differences in the sialopolysaccharideconcentration associated with groups A and C streptococci.Given the reported observations of lectin interactionswith various bacteria, the most convenient means oftesting lectins as a diagnostic tool is generallyassociated with the application of isolated bacterialcolonies. The direct detection of a particular bacterialagent in a clinical specimen with the use of a lectinassay may be difficult to attain. This is due to thepotential for a relatively wide variety of carbohydratesubstrates to be available for lectin interaction in agiven clinical specimen. Inoculation of clinical specimensonto tissue culture cells has permitted a specific andsensitive means to detect herpes simplex virus with useof fluorescein-conjugated H. pomatia lectin [112]. Theusefulness of lectins in clinical microbiology isassociated with an extremely broad spectrum of advantagesthat render them of value in routine diagnostic procedures.Many of these diagnostic procedures associated withlectins are relatively inexpensive and can be rapidlycompleted. Lectins can also be used for light and electronmicroscopic investigations [113]. Some of the significantadvantages are (1) their stability, (2) their activity invery small concentrations, (3) the commercial availability

of many leetins, and (4) their ability to probe subtlesurface structural differences between various isolates[22]. The cost-effectiveness of these unique naturallyoccurring cytochemical-histochemical tools furtheremphasizes their value as diagnostic reagents for theclinical microbiologist. It was previously suggested thatbecause of the binding specificity of 165

lectins, they may be able to replace immune sera indetecting various carbo

hydrate residues on microbial cells and, by extrapolation,particular micro

organisms [19]. This chapter has emphasized that lectinsare versatile re

agents that can be applied in certain instances to (1)provide definitive

identification, (2) characterize a strain of a particularorganism, and (3)

function as epidemiological markers. The effect ofstandard methodology and contemporary emphasis on

molecular biology technology associated with monoclonalantibodies and

nucleic acid probes has, in part, diverted attention fromthe potential of

lectins in applied and clinical microbiology. Furthermore,the unique carbo

hydrate specificities of lectins in comparison with thespecificity attained

with the use of monoclonal antibodies or nucleic acidprobes may also be a

factor considered by various investigators. In contrast, anexamination of

an increase in patented processes on diagnostic uses oflectins for the last 5

years, clearly indicate a significant increase in the useof lectins in scientific

research and in clinical microbiology. The emphasis ofthis chapter is on the application of lectins derived

from plant seeds, pulp, bark, root, bulb, and invertebrateextracts for the

identification and characterization of bacteria. The useof lectins obtained

from certain bacteria may also be of value as diagnostictools for clinical

microbiology. The interaction of lectins from Pseudomonasaeruginosa,

for example, may be of value for the identification ofvarious E. coli strains

[114,115]. The mannosephilic hemagglutinin of P.aeruginosa is reported

to agglutinate the cells of the enteropathogenic E. coli0128: B12 and

086 : B7. The binding of the lectin was shown by specificagglutination of

the E. coli strains by hemagglutination tests [ 114]. Theuse of this bacterial

lectin for the typing of these potential enteropathogenswas considered to

have certain distinct advantages [115]. Although lessspecific than antibod

ies, the bacterial lectin probe can be conjugated with aperoxidase signal

[115]. Furthermore, in the realm of clinical microbiology,certain lectins

may also have antibacterial activity [116], and they havebeen considered as

carriers of chemotherapeutic agents [12]. There are now awide variety of lectins available in unconjugated as

well as conjugated forms from several commercial sources.Furthermore,

panels of lectins under the trade name of Taxonolectinsare commercially

available for the identification and characterization ofbacteria from E-Y

Laboratories, San Mateo, California. The published reviewson the diagnostic applications of lectins in mi

crobiology as well as the commercial availability of arelatively wide source

of lectins should provide ample opportunities to furtherassess the applica

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5 Lectin

16. Geisow M. Shifting gear in carbohydrate analysis.Biotechnology 1992; 10: 277-280.

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18. Richardson MD, Kearns MJ, Smith H. Differentiation ofextracellular from ingested Candida albicans blastosporesin phagocytosis tests by staining withfluorescein-labelled concanavalin A. J Immunol Methods1982; 52:241-244.

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25. Calderone RA, Linehan L, Wadsworth E, Sandberg AL.Identification of C3d receptors on Candida albicans.Infect Immun 1988; 56:252-258.

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C3d receptor, isolated by using a monoclonal antibody.Infect Immun 1988; 56: 1981-1986.

27. Bramley TA, Menzies OS, Williams RJ, Kinsman OS, AdamsDJ. Binding sites for LH in Candida albicans: comparisonwith the mammalian corpus luteum LH receptor. JEndocrinol1991; 130:177-190.

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29. Critchley lA, Douglas LJ. Role of glycosides asepithelial cell receptors for C. albicans. J GenMicrobiol1987; 133:637-643.

30. Sandin RL, Rogers AL, Patterson RJ, Beneke ES. Evidencefor mannosemediated adherence of Candida albicans to humanbuccal cells in vitro. Infect Immun 1982; 35:79-85.

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32. Jimenez-Lucho V, Ginsburg V, Krivan HC. Cryptococcusneojormans, Candida albicans, and other fungi bindspecifically to the glycosphingolipid lactosylceramide(GAI{j1-4Glq31-1Cer), a possible adhesion receptor foryeasts. Infect Immun 1990; 58:2085-2090. 33. BlackwellCC, Thorn SM, Weir DM, Kinane DF, Johnstone DF.Host-parasite interactions underlying nonsecretion ofblood-group antigens and susceptibility to infections byC. albicans. In: Lark DL, ed. Protein-carbohydrateinteractions in biological systems. London: Academic Press,1986:231-233. 34. Maisch PA, Calderone RA. Role of surfacemannan in the adherence of Candida albicans to fibrinplatelet clots formed in vitro. Infect Immun 1981;32:92-97. 35. Ray TL, Digre KB, Payne CD. Adherence ofCandida species to human epidermal corneocytes and buccalmucosal cells: correlation with cutaneous pathogenicity. JInvest Dermatol1984; 83:37-41. 36. Rotrosen D, Edwards JEJr, Gibson TR, Moore JC, Cohen AH, Green I. Adherence ofCandida to cultured vascular endothelial cells: mechanismsof attachment and endothelial cell penetration. J InfectDis 1985; 152:1264-1274. 37. Klotz SA Penn RL. Multiplemechanisms may contribute to the adherence of Candidayeasts to living cells. Curr Microbiol1987; 16:119-122.38. Segal E, Lehrer N, Ofek I. Adherence of Candida

albicans to human vaginal epithelial cells: inhibition byamino sugars. Exp Cell Bioi 1982; 50:13-17. 39. MiyakawaY, Kuribayashi T, Kagaya K, Suzuki M, Nakase T, Fukazawa Y.Role of specific determinants in mannan of Candida albicansserotype A in adherence to human buccal epithelial cells.Infect Immun 1992; 60:2493-2499. 40. Hasenclever HR,Mitchell WO. Antigenic studies of Candida. I. Observationof two antigenic groups in Candida albicans. JBacteriol1961; 82:570-573. 41. Kagaya K, Miyakawa Y,Fujihara H, Suzuki M, Soe G, Fukazawa Y. Immunologicsignificance of diverse specificity of monoclonalantibodies against mannans of Candida albicans. Jlmmunol1989; 143:3353-3358. 42. Kobayashi H, Shibata N,Suzuki S. Evidence for oligomannosyl residues containingboth {j-1,2 and a-1,2 linkages as a serotype A-specificepitope(s) in mannans of Candida albicans. Infect lmmun1992; 60:2106-2109. 43. Shibata N, Arai M, Haga E, KikuchiT, Najima M, Satoh T, Kobayashi H, Suzuki S. Structuralidentification of an epitope of antigenic factor 5 inmannans of Candida albicans NIH B-792 (serotype B) andJ-1012 (serotype A) as (j-1,2-linked oligomannosylresidues. Infect Immun 1992; 60:4100-4110. 44. Warnock DW,Speller DCE, Day JK, Farell AJ. Resistogram method fordifferentiation of strains of Candida albicans. J ApplBacteriol1979; 46:571578. 45. Odds FC, Abbott AB. A simplesystem for the presumptive identification of Candidaalbicans and differentiation of strains within the species.Sabouraudia 1980; 18:301-317. 46. Kohler G, Milstein C.Continuous cultures of fused cells secreting antibody ofpredefined specificity. Nature 1975; 256:495. 47. Tojo M,Shibata N, Kobayashi M, Mikami T, Suzuki M, Suzuki S.Preparation of monoclonal antibodies reactive with(j-1,2-linked oligomannosyl residues in thephosphomannan-protein complex of Candida albicans NIHB-792 strain. Clin Chern 1988; 34:539-543.

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6

Lectin-Leishmania Interaction

R. L. JACOBSON The Hebrew University-Hadassah MedicalSchool, Jerusalem,

Israel

I. INTRODUCTION

Leishmania are digenetic (heteroxenous) parasitic protozoaof humans and

animals that are found alternatively as flagellated, motilepromastigotes

and paramastigotes in the alimentary tract of phlebotominesandflies, or as

obligate intracellular aflagellate amastigotes in thephagolysosomes of host

macrophages. The genus Leishmania is of the orderKinetoplastida, family

Tryposomatidae. There are over 20 designated species andseveral unnamed

species grouped into two subgenera [1] (Table 1). Thesubgenus Leishmania

has been further divided into three complexes, the L. (L.)donovani com

plex, for which development in the sandfly vector isrestricted to the midgut

and foregut of the alimentary canal (suprapylaria), andthose Old World

species outside this complex (e.g., L.(L.) major) and the

New World com

plexes L.(L.) mexicana and L.(L.) hertigi. The othersubgenus is L.(Vian

nia) braziliensis and is characterized by prolific andprolonged phases of

development in the hindgut of the sandfly vector, withlater migration of

the flagellates to the midgut and foregut (peripylaria).This genus is re

stricted to the American tropics and subtropics. Othermethods, such as

isoenzyme profiles, serotyping, and kinetoplast DNA buoyantdensities,

have been used in attempts to characterize the differentspecies groups, and

each method has its own criteria. Lectins, when used astaxonomic tools,

follow more closely the broad pathological conditionscaused by the para

sites than do some other methods. The leishmaniases are agroup of pathological conditions ranging from

a simple cutaneous lesion caused, for example, by L.major, through to the 191 Table 1 Taxonomy of the genusLeishmania Subgenus Leishmania Viannia Source: Updatedfrom Ref. 61. Complex L. donovani L. tropica L. majorL. aethiopica L. mexicana Not pathogenic to humans L.hertigi L. braziliensis L. guyanensis UnassignedSpecies L. archibaldi L. chagasi L. donovani L.infantum L. killicki L. tropica L. major L. aethiopicaL. amazonensis L. garnhami L. mexicana L. pifanoi L.venezuelensis L. arabica L. gerbilli L. turanica L.aristidesi L. enriettii L. hertigi L. deanei L.braziliensis L. peruviana L. guyanensis L. panamensisL. /aisoni disfiguring diffuse cutaneous leishmaniasis (L.aethiopica) and mucocutaneous leishmaniasis (L.braziliensis) and, finally, to the fatal visceralleishmaniasis (L. donovani). There is variation withinspecies, as L. tropica is both cutaneous and occasionallyvisceralizing and L. donovani is found in a condition

known as post-kala-azar dermal leishmaniasis. Both formsof the parasite invade macrophages and, as primary contactis at the cell surface level, it is this host-parasiteinterface that has been extensively studied. Cleardifferences in the surfaces of the parasite have beenobserved among strains that are otherwise closely related.Soluble and membrane-bound proteins linked tooligosaccharides and termed glycoconjugates, constitutesome of the major components of surface material. Specificprobes, such as lectins, have added extensively toknowledge of the cell surface carbohydrates and are beingused to study their function. Released and cellmembrane-bound carbohydrate determinants have

both been cited as being important in the biology ofleishmania! parasites.

Surface carbohydrates of leishmania! promastigotes areimportant for at

tachment to the vertebrate host macrophage [2-4] and thesandfly digestive

tract [5]. It has also been proposed that they play a rolein the protection

and survival of the parasite at the onset and during theinfection of the

host macrophage [6, 7] and against the digestive tractenzymes of the sandfly

vector [8]. The antigenic determinants on the surface ofthe parasite are oligosac

charides, but the nature of their structural associationwith glycoproteins,

glycolipids, or other molecules is still beinginvestigated. Lectin probes

have been used to determine the presence of carbohydratereceptors on

Leishmania. Dwyer [9] was the first to report the bindingof lectins to

saccharides on the surface of promastigotes. Agglutinationtechniques and

fluorescein-labeled lectins have revealed that othercarbohydrate residues

are on the surface of the parasites as well as in theexcreted glycoconjugate.

Differences have been found in the configuration andquantity of the carbo

hydrate moieties among species and strains [10-12]. Thesurface membrane carbohydrates of different strains ofLeish

mania have been examined with lectins [13-16]. Theseauthors showed vari

ation in the carbohydrate determinants of the differentspecies and have

revealed interand intraspecific differences in promastigotesurface carbo

hydrate moieties. Glucose or mannoselike residues are foundon the sur

faces of all Leishmania, but in greater abundance in theL. tropica strains

[13]. Five L. tropica strains were tested forlectin-mediated agglutination

with seven lectins and only ConA, RCA, and SBA, (seeChapter 1 for ab

breviations) were positive [17]. Galactose residues asreflected by lectin agglu

tination, are common to all species, but to a smallerextent in L. donovani

and L. mexicana strains. Eleven strains of L. major wereinvestigated, and

it was reported that the surface components and strains ofthis species

display marked heterogeneity [18]. In retrospect, theseearlier studies of surface and released carbohy

drates used material harvested at a given point during the

parasite's growth

cycle, but did not investigate the infective potential ofthe parasites. It

has since been shown that stationary-phase (S-phase)promastigotes, which

agglutinate with lectins differently from exponential phaseparasites, are

more infective to mice [19]. The lectin RCA-I (RCA 60 ),which is specific for

galactose ({jGal) and, but less so, forN-acetyl-o-galactosamine (GalNAc),

agglutinated fewer cells from the S-phase of L. donovani.Promastigotes

from this phase gave a greater parasite burden in the liverof the infected

mice [19]. The lectin of Arachis hypogaea (peanut; PNA),which is specific

for Gal{j-1,3GalNAc > {jGal, was used as a marker to showthat S-phase promastigotes that did not agglutinate withthis lectin caused larger lesions in infected mice [20].These promastigotes are the infective forms and the finalstage of metacyclogenisis. More recently, alipophosphoglycan (LPG) has been defined, consisting of atripartite molecular structure [21]. This LPG consists of apolymer of repeating phosphorylated saccharide unitsattached by a carbohydrate core to a novel lipid anchor.The prominent feature of LPG is the polymer of 16phosphorylated disaccharides of [P0 4 -+ 6Gal~1 -+ 4)Mana1]units. In L. major, the analogous portion is composed of aseries of small oligosaccharides, consisting of severalcommon hexoses and the pentose arabinose [22]. Thestructurally related molecule, phosphoglycan, is shed bypromastigotes in vitro and is present in spent culturemedium [23]. It has been suggested that LPG is amultifunctional molecule [21]. There is evidence that inthe natural habitat of the promastigote, the sandfly gut,the shed carbohydrates are found as a gellike matrix inboth the midgut and the cardia in histological sections[24] and by immunocytochemical analysis [25]. The releasedglycoconjugate also enhances the survival of foreignpromastigotes in the indigenous host, indicating a

vector-specific function of the shed material [26]. II.LECTIN-MEDIATED AGGLUTINATION A. Single Strains The factthat lectins could agglutinate promastigotes and be used todetermine the presence of carbohydrate receptors onLeishmania was first described in 1974 [9]. In this firstreport, the binding of lectins to saccharides on thesurface of promastigotes was described in a single species,L. donovani, with three lectins. This author was able tofind evidence of glucose or mannose andN-acetylgalactosamine randomly distributed on the surfaceof the parasite using the lectins ConA andphytohemagglutinins M and P. There have been additionaldescriptions of single isolates and their reactions tovarious lectins, using a variety of techniques. Theseincluded agglutination with five lectins (ConA, RCA, WGA,SBA, and PHA-P) of different stages of growth of L.braziliensis [11]; an agglutination and electronmicroscopic study of horseradishperoxidase (HRPO)-labeledConA on the surface of L. braziliensis guyanensis [27].Another study reported agglutination tests with 31 lectinsof which 17 reacted with the promastigotes, andfluorescent microscopic examination of 6 fluoresceinisothiocyanate (FITC)-labeled lectins on the surface ofL. tropica [15]. The original work on L. donovani clone1-S, Cl 2 , was expanded with agglutination tests withfive lectins (ConA, PHA-P, SBA, WGA, and LOTUS) and use of[ 3 H]ConA

Lectin-Leishmania Interactions 195

and HRPO-ConA [10]. The results of these studies showedthat, whereas

mannose and glucose and galactose were common to allpromastigotes,

GalNAc, GlcNAc, and fucose were not found on the New Worldstrains. It

was also noted that three types of agglutination occurred,flagellar-flagel

lar, flagellar-body, and body-body, indicating that theoligosaccharides

are widely distributed on the surface of leishmania! cells[9,27]. It has also

been reported that there are indications that sugarssimilar to a-o-mannose

and a-D-glucose, o-galactose, N-acetylgalactosamine,N-acetylglucosam

ine, and a-L-fucose are present on the surface of variousLeishmania species

[28]. Mannose or glucose and galactose appear to be commonmembrane

lectin receptors for most species of Leishmania; however,the literature is

somewhat equivocal concerning the other sugars. Theforegoing studies

were based on single isolates of four species.

B. Comparison of Strains and Species

A comparison of five isolates of L. tropica from Iran andL. d. chagasi, L.

m. mexicana, and L. m. amazonensis with seven lectins(ConA, RCA,

SBA, WGA, PHA, PWM, and LOTUS) indicated some speciesspecificity

[17]. All strains tested were positive with ConA. All fivestrains of L.

tropica were positive with RCA and SBA, but were negativewith the other

lectins. Leishmania was positive with RCA and WGA; L. d.chagasi was

positive for RCA; and L. m. amazonensis was negative exceptfor the

mannose-glucose-specific lectin. It was then suggestedthat lectins could be

used for taxonomic determination of species. Lectinagglutination of promastigotes was linked to an existingsero

type system [13] and was also suggested as an additionaltaxonomic tool

[13,14,16]. Table 2 is an attempt to tabulate all thelectin agglutination

data currently available for which the strains arereasonably well docu

mented, and the more popular lectins are available to otherresearchers. In

Table 2, the many variations of scoring results byindividuallectinologists

have been reduced to the simple formula of "+" for strongagglutination,

"::1:" for weak results, and "-" for nonagglutination. Themain conclusions

that can be drawn from this body of work is that theMiddle-Eastern strains

of cutaneous leishmaniasis, caused by either L. major or L.tropica, were

nearly always strongly agglutinated with SBA (GalNAc) andUEA-I (Fuc)

or UEA-II(GlcNAc} 2 • In the New World, the one speciesthat has a clearly

defined lectin profile is L. m. pifanoi [14]. The onestrain of this species,

LRC-L90, that was tested, was agglutinated by ConA, RCA,PNA, SOJ,

SOH, UEA, but not with PHA or AAP [14]. No other New Worldspecies

reflected such a diverse and rich pattern ofoligosaccharides on their sur

faces. The Old World species L. aethiopica would appear tobe at the Table 2 Lectin-Mediated Agglutination ofLeishmania Species ConA PNA o-Man RCA120 {3-o-GalSpecies Designation Ref. o-Gle {3-o-Gal 1-3GalNAc OldWorld L. dono1-S 10 + nd nd vani l-S,3S,L-51 ,L V-13914 + + + L. do 16 + nd L. doT 16 + + ndLRCL-52,L-133,L-210 13,34 + ± ±I+ L-133 16 + + nd

Sudanese 43 + + + L. d. LV-140 14 + + infantum K26316 + + nd LV9 54 + + + L. major L V252,L V -253,L-3814 + + nd (USSR) LRC-L38 18 + + + Neal-P 18 ± ++ L. major Ko,Ha,Schwe,Ne,Ro,Ve 14 + + + (M. East)LRCLl37 ,L287 ,L288,L31 13,18,34 ± + ±/+ L306,L464,L505,L23 18 + + + L223 16 + + nd L251,L137 54 + + +L. major L448,L461 18 + + -I+ (Kenya) Ll19 18 ± +± L. tropica LV-249,LRC-L39 14 + + + LRCL-32,L-289,L-3913,34 + + + LV556,L-32 16 + + nd L-36 54 + + LV-116 + + nd L. aethioLV-1,LV-15,LV-24,LV-26 14 + + picaLRC-L134,Ll47 13,34 ±I+ + ± New World L. b. brazili11+ + nd ens is M2903 54 + + ± M2903,LRC-L 77 14 + +L.b. panaWR120 54 + + mens is L.m. LRC-L94 13 + +mexicana LRC-L94,M379 14 + + Ll1 16 + + nd Lll 54± + L.m. LRC-L259 16 + + nd amazoLTB0016 54 + ±nensis H21,M1696 14 + + L.m. LRC-L90 14 + + + pifanoiKey: -, no reaction; ±, weak reaction; +, strong reaction;nd, not done. 197 Lectin SJH PHA WGA SBA (jo-GalNAcULEX-1 ULEX-11 o-GalNAc (o-GlcNAch AAP

o-GalNAc (jo-G a! L-Fucose (o-GlcNAc) 2 L-Fucose NeuNAco-GlcNAc + nd nd nd + + nd nd nd nd ± ± nd nd nd+ ± nd nd nd nd nd nd + + + nd nd nd nd nd nd nd+ + + nd nd + + ± nd nd + nd nd nd nd nd nd + nd+ nd nd nd nd + nd nd nd nd nd + + + nd + nd ±I++ nd + nd + + nd nd nd nd + ± + nd nd + nd ndnd nd nd + nd + nd nd nd + nd + + nd + + + ++ nd nd + nd nd + + + + nd nd + nd nd nd nd nd nd+ nd nd nd nd -I± nd nd nd nd nd nd nd ± nd ndnd nd nd nd nd ± nd nd nd nd nd ± nd nd nd ndnd nd nd ± nd nd nd nd nd nd nd nd nd nd ± ndnd nd nd nd nd nd nd + + + nd opposite end of thespectrum, as promastigotes of some strains of this speciesare strongly agglutinated only by RCA-II and very weakly soby ConA, PNA, SOJ, and SBA [13,14,16,28]. The questionmust arise whether enough lectins have been used to form apanel of lectins that could separate the main complexes,if not individual species. Thirty-one lectins were used toexplore the surface membranes of L. tropica and Crithidia/ucillae (an insect protozoan) [15], and 23lectins wereused by the same group of researchers in an attempt toidentify and classify different species [16].Notwithstanding that some of the strains used have beenreclassified as different species [i.e., L. major (simplecutaneous leishmaniasis) is now a species distinct from L.tropica and L. infantum (visceral leishmaniasis with ananimal reservoir) is distinct from L. donovani (man theonly reservoir)], these authors concluded that lectinagglutinations added to our general knowledge of the"calling card" of the leishmaniae, but could not be used

definitively for taxonomic classification [29]. In my ownlaboratory, six lectins (ConA, RCA, PNA, SBA, UEA-1, andUEA-11) have been found to be the most useful in membraneagglutination analysis. We have also used LAA todifferentiate L. arabica, a rodent parasite found inPsammomys obesus, from human isolates of L. major fromTurkestan, where the reservoir host is Rhombomys opinus.This lectin gave identical agglutination results for L.arabica from Saudi Arabia and for L. major strains inIsrael, whether isolated from humans or the reservoir hostP. obesus. As there is such variation among the L. majorstrains and isolates, we decided to investigate 11 suchstrains with a standard panel oflectins [18]. 1. LectinSpecificity of 11 Strains of Leishmania major The resultsof the lectin-mediated agglutination tests are presenteddiagrammatically in Figure 1. Agglutination; shown on thevertical axis, is graded from 0.0 (no agglutination) to4.0 (all parasites agglutinated). Little uniformity wasseen in the lectin agglutination profiles of the strains,even those of the same serotype. A lectin specific for{3-1-o-galactose, RCA-II, agglutinated all the strainsstrongly. Concanavalin A, specific for o-mannose ando-glucose, caused different degrees of agglutinationamong the strains. Strain LRCL119 and the P strain wereweakly agglutinated, whereas strains LRC-L23 and LRC-L38reacted strongly. The PNA lectin, with an affinity for thegalactose-N-acetylgalactosamine dimer, caused moderateagglutination in most strains, but only feeblyagglutinated strain LRC-L119 and had no effect on strainLRC-L461. The SBA lectin, specific forN-acetylgalactosamine, also agglutinated most strainsmoderately, but did not agglutinate strain LRC-L38. LectinUEA-1, specific for L-fucose, agglutinated G) '0 c c 0; (11 c .. :I en en <1: >< G) '0 c c 0 ..(11 c .. :I en en <1: ,.... CD 'It tO C") 0 CD 0..M 'It &tl RCA co Q. a) Q) 'It .... C") U) ... ~ ConA I 4 1 Ulex-1 co Q. co Q) ..... .... ,.... U) "" IJ) (")co Q. co (J) OCDONM V.,...(")U) MOCDONM ""'~"'"MID C")'It &.n 'It ,... 'It .M o::t IJ) 'It .,... 'It LeishmaniaReference Centre (LRC) strain number SBA ,.... CD 'It IJ)C") CO Q. CO (J) .,... .,... C") 0 CD 0 N C") 'It .,...(") CD .... (") 'It IJ) 'It .... 'It ,.... CD 'It IJ) C")CO Q. CO Q) M 0 CD 0 N C") 'It .,... C") CD 'It IJ) 'It.,... 'It Ulex-11 o::t ..n M co c.. co en M 0 <0 0 N C")... ., Leishmania Reference Centre (LRC) strain number "'... Figure 1 Lectin-mediated agglutination of 11 strainsof L. major. Agglutination index; scale range: 0, noagglutination; 4, all cells agglutinated. (Modified fromRef. 18.) s· I ,.... ~ ;;;· :r 3 AI :I 6'' ::1~ ... AI !l i5' ::1 <I> .... '-'1 '-'1 strains

LRC-L137 and LRC-L23, but reacted only weakly, or not atall, with the other strains, and UEA-11, specific fordi-N-acetyl-o-chitobiose, failed to agglutinate strainLRC-L38 and the P strain, but agglutinated all otherstrains moderately. When promastigotes were incubated withfluorescein-labeled lectins, we were unable to detect anylabeling, as monitored by microscopy, with those lectinsthat had also failed to agglutinate them. Inhibition ofthe lectins with the appropriate specific sugars revealedsome unexpected findings. Of the 11 strains agglutinatedby ConA, only 2, strains LRC-L461 and LRC-L119, were notinhibited by a-1-o-mannopyranoside, but were with glucose.Lactose failed to inhibit UEA-11 agglutinin in four outof eight strains checked, LRC-L306, LRC-L464, LRC-L505,and LRC-L31. Two of these strains, LRC-L464 and LRC-L505,were inhibited from agglutinating by chitobiose, whereasthe other two were not. All 11 strains of L. major testedhave {3-1-o-galactose moieties on their surface membranes,as evidenced by Ricinus 120 binding. These results were inagreement with previous studies [13,14]. Concanavalin A hasalso been reported to agglutinate all species ofLeishmania [13,14,16]. This usually indicates the presenceof mannose and glucose residues on the surface membrane.The saccharide, a-1-o-mannopyranoside, failed to inhibitConA agglutination of strains LRC-L119 and L461, but wasinhibited by glucose. These two Kenyan strains areradically different from other strains of L. major.Strain LRC-L119 does not release glycoconjugates detectableby our methods [4;30] and LRC-L461 is one of severalstrains of L. major that produces serotype B EF [31-33].The lectin UEA-1, which is specific only for L-fucoseresidues, reacted diversely with the 11 strains. StrainsLRC-L38 and P, two lines derived from an original isolate,from a Rhombomys opinus caught in Turkestan, and both EFsubserotype ~. failed to agglutinate and, therefore,appear to lack L-fucose as terminal residues. StrainsLRC-L448 and LRC-L461, both from Kenya and subserotype ~B2 and B 2 , respectively, also lacked L-fucose. Very weakagglutination occurred in the other Kenyan strain,LRC-L119, and the Israeli strain LRCL31, a strainrepresenting the EF subserotype A 1 B 2 • Conversely,strain LRC-Ll19, as reported [34], was not agglutinatedby an UEA lectin that could be inhibited by lactose, butnot fucose. This anomaly may have been due to the potencyof the reagents used; as both UEA-11, specific for GalNAc,and UEA-1 did agglutinate cells from this strain(LRC-L119). The other Israeli strains showed variousreactions to UEA-1, from weak in strain LRC-L306subserotype A 1 , to very strong in strain LRC-L23,subserotype A 4 • Strain LRC-L38 and other Turkestani

strains of L. major also lack L-fucose residues [14].This might explain some of their differences from strainsfound in the Middle East, as was suggested on clinicalgrounds [35]. 201

Strains LRC-L119 and LRC-L461 also appeared to lackgalactose (as shown

by PNA agglutination reactions) or have considerablyreduced amounts of

it when it is coupled to part of the N-acetylgalactosaminedimer. However,

both have sufficient {3-1-D-galactose andN-acetylgalactosamine to react

with RCA-II and SBA agglutinins. The promastigotes ofthese two strains,

therefore, seem to have lectin profiles that differconsiderably from the

other L. major strains (see Fig. 1). Strain LRC-L38 andthe P strain, both derived from the same isolate,

were maintained in Jerusalem and London, respectively, formany years.

Although these two lines have identical enzyme and serotypeprofiles (zy

modeme LON 1) and EF subserotype (A 4 ), their reactionswith lectins are

different. Promastigotes of LRC-L38 appeared to lackGalNAc on their

surface membrane, whereas the promastigotes of the Pstrain bound well

with SBA. Conversely, strain LRC-L38 bound strongly withConA, show

ing an abundance of mannose and glucose residues, and Pstrain showed

very weak agglutination with this lectin. There is alwaysthe possibility of a

human error, but it is unlikely here, because the enzymeprofiles are identi

cal and, more importantly, both had A 4 subserotypes.Therefore, this shows

clearly that the strains have become different in theirreactivity to some

lectins, even though they are now grown under the sameconditions. The strains LRC-L306 and LRC-L31 differ inboth enzyme profile and

serotype, yet their lectin profile is very similar.Although both are readily

agglutinated by UEA-11 lectin, neither reaction wasinhibited by the addi

tion of lactose or chitobiose, indicating that both havean unusual carbo

hydrate configuration on their surface membrane. If acontaminant was

present in the UEA-11 lectin it would be very difficult toascertain which

saccharide residue was responsible for the agglutinationreaction. Some

UEA-11 commercial reagents may agglutinate cells with asaccharide chain

of L-Fuca1,2Gal{31,4GlcNAc, but considering the weakerreactions these

two strains gave with other lectins, this configurationseems unlikely. This study showed that L. major strainshave a diversity in their lectin

specificities, as they do in their serotypes. There is nodirect evidence that a

universal increase in one carbohydrate denotes the decreasein another.

Nor does there appear to be a direct correlation betweenthe carbohydrate

topography expressed on the surface membranes and releasedcarbohydrate

moieties as determined by lectins. The one lectin thatseems to indicate a

species specificity is SBA, which is specific for GalNAc,and the only excep

tion was the strain LRC-L38, which was agglutinated byneither UEA-1 nor

UEA-11. This diversity of lectin-mediated agglutinationindicates that no

single strain typifies this species with respect to itssurface and released

carbohydrates. These diversities were so pronounced forthese 11 strains that it was decided to investigatewhether different-growing conditions could modulate thesurface carbohydrates expressed by the promastigotes [36].2. Surface Carbohydrate Expression of Leishmania major inTwo Media The two media chosen were the biphasic (Novy,MacNeal, and Nicolle's) (NNN) medium, rich in bloodcomponents and Schneider's medium (SDM), a semidefined allliquid medium, in which inactivated fetal calf serum servesas a protein source. A cloned line of L. major, strainLRC-L137, was initiated and a duplicate series of culturetubes were inoculated with a standard dose ofpromastigotes from the S-phase of growth, washed free ofmedium components,. in phosphate-buffered saline (PBS), pH7 .2. Promastigotes were tested daily for their ability toreact with the panel of lectins [36]. In the NNN mediumbetween day 3 and 4, there was an increase in the numberof promastigotes that could be agglutinated by the lectinsRCA, PNA, SBA, and UEA-I (Fig. 2), followed by a decreaseuntil the end of the growth cycle. In SDM, thepromastigotes manifested trends similar to those in NNNfor the lectin UEA-I, but with PNA, RCA, and SBA there waseither an increase or agglutination remained the sameduring the stationary phase (see Fig. 2). In both media,there was some increase in the number of cellsagglutinated during the growth cycle when they wereincubated with ConA and UEA-11. As a population ofcells, the promastigotes grown in NNN medium to stationaryphase, showed a clear tendency to lose their ability tobind lectins that were specific for galactose,N-acetylgalactosamine, and fucose. Although cells grown ina semidefined medium showed a similar trend for the

fucose-specific lectin (UEA-1), they were bound more tothose lectins specific for either {3-1-galactose or GalNAc(PNA, RCA, and SBA) during the stationary phase than theywere during the exponential phase. This clearly indicatesthat the cell surface receptors can be affected by thenutrient contents of their surrounding medium, and thatthe promastigote population adapts either by selection, orchanges metabolically with the digestive tract of thesandfly [37] or with the extracellular milieu of themammalian host. It has been suggested that the uptake ofthe natural carbohydrate diet of the sandfly in the wildmay profoundly affect the ability of the parasite tosurvive and be transmitted to the mammalian host [38]. Inanother study, the presence of lectinlike receptors wasreported in Phlebotomus papatasi homogenates, the mainvector of L. major [39]. This lectin activity was

Lectin-Leishmania Interactions 203 2

)(

Q)

-o 0 c 4

c 3 0

c 2

c I

:::1 0

C>

C>

<t

Figure 2 Daily changes of lectin-mediated agglutination ofpromastigotes of L.

major (LRC-L137) grown in two media: filled circle, NNN;open circle, SDM.

Agglutination index; scale range: 0, no agglutination; 4,all promastigotes aggluti

nated. (From Ref. 36.)

inhibited by only two oligosaccharides, trehalose andturanose, for which

there is no specific plant lectin analogue. Trehalose isthe main disaccharide

found in the hemolymph of many insects as well as beingwidely distributed

in bacteria, yeasts, and fungi, whereas turanose can befound in the disac

charide fraction of honey [40]. This would suggest thatthese inhibiting

saccharides are either of insect origin or are taken up aspart of the sandfly

carbohydrate diet [41]. The sequentially changingconfigurations of carbo

hydrate moieties that were found in L. major may well bein response

to the presence of these lectinlike receptors in the headsand midguts of

sandflies. Although agglutination of promastigotes by thelectins has increased

our knowledge of the carbohydrate residues of the cells,the use of labeled

lectins has permitted a more intensive investigation ofthe surface oligosac

charides. Ill. LABELED LECTINS AND SURFACE TOPOGRAPHY Theuse of labeled lectins, whether conjugated with fluorescentdyes, ferritin, horseradish peroxidase, or radioactivelabels, has enabled various workers to map the surfacemembranes of leishmania! promastigotes. A. FluorescentLabels There have been two main tools used to detect thepresence of fluorescent labels on the surface ofpromastigotes: the fluorescence microscope and thefluorescence-activated cell sorter (F ACS). The microscopehas been useful in detecting the region of the lectinreceptors and visualizing some receptors that gavenegative reactions in agglutination tests. The FACS, withits argon laser, has been used to show kinetic labeling indifferent populations of cells, as well as revealing

insufficient lectin receptors to cause agglutination [42].1. Fluorescence Microscopy In an early study [15], astrain of L. tropica, (unfortunately unidentified), wascompared with Crithidia /ucillae, a nonpathogenic insectflagellate. Rather surprisingly, only fluorescein-RCAandfluorescein-UEA labeled the parasites, whereas labeledPEA, LCA, and LAO were negative. This is rather anunusual finding, as these latter lectins are specific formannose and glucose, which are common to almost everystrain of Leishmania ever tested. A Sudanese strain ofL. donovani was comprehensively studied at both theamastigote (intracellular) and promastigote (extracellular)stages [43]. The six FITC-labeled lectins used were ConA,PNA, WGA, RCA, SBA, and LOTUS. In this elegant study,there was a clear distinction between the stages of theparasite according to their lectin binding. Whereas LOTUSand SBA were negative, indicating the absence of GalNAc andfucose, PNA was positive only for promastigotes, and WGAwas positive only for amastigotes. Another importantdifference indicated in this work was that, whereasFITC-ConA binding on promastigotes was inhibited by 0.8 mMo-mannose, only 10 mM a-methylmannopyranose would inhibitthe FITCConA binding to amastigotes. The authors alsoshowed that the WGA binding on amastigotes was decreasedby GlcNAc and not sialic acid, even after sialidasetreatment. In a more recent study [44], three Indianstrains have been investigated with eight FITC-lectins,including ConA, DBA, PHA-P, PNA, RCA-II, SBA, UEA-I, andWGA. The three strains investigated were a visceral L.donovani, a post-kala-azar dermal leishmaniasis L.donovani, and a cutaneous L. tropica. The FITC-labeledSBA, PNA, and WGA failed to

label the visceral leishmanial strain, whereas the othertwo strains were

labeled by all the lectins. The authors point out thatthese results are not in

agreement with their previously published results usinglectin agglutination

tests [45].

2. Flow Cytometry

The other use of FITC-lectins has been with studies ofpopulations of

parasites using flow cytometry with fluorescence-activated

cell sorters

(FACS). Both the kinetics of changing surface carbohydratesduring the

sequential growth of a parasite population and comparisonsof different

species have been actively pursued in my laboratory. Wehave made a comparison of the FITC-lectin receptors on sixL.

major strains, three isolated in Israel (LRC-L306, -L464,and -L23), two

from Turkestan (LRC-L38 and P strain), and one from theSinai Desert

(LRC-L505). The FITC-lectins used were RCA, ConA, PNA,WGA, and

SBA. All the parasites were grown in Schneider's definedmedium, with

lOOJo fetal calf serum, until the stationary phase.Promastigotes were har

vested, washed three times in PBS, pH 7 .2, and fixed in1% formaldehyde

in PBS, with 2% glucose and 0.1% sodium azide. TheFITC-lectins have

been standardized in our department, and are used asfollows: FITC-PNA

and FITC-WGA, 20 p.g/ml; FITC-SBA, 10 p.g/ml; FITC-RCA, 5p.g/ml;

and FITC-ConA, 0.6 p.g/ml. The results showed markedheterogeneity

among the strains and little correlation withlectin-mediated agglutination

results [36,42] (Table 3 and see Fig. 1). Strains thatwere not agglutinated,

such as LRC-38 by SBA, bound well with the FITC-labeledlectin. Further

more, FITC-WGA labeled between 20 and 40% of all the cellstested in this

series and, although there are almost no reports of thislectin agglutinating

promastigotes, the presence of GlcNAc has been confirmedby other meth

ods [22]. The use of flow cytometric techniques allowedus to measure the changes

of the carbohydrates on the surface of the parasites duringthe various

phases of growth [36,42]. Although very little variationwas found in FITC

ConA and FITC-RCA binding during the growth of strainLRC-L544 (a

freshly isolated Israeli L. major), indicating theconstancy of mannose and

galactose, other lectins showed definite changes. TheFITC-SBA labeled

twice as many cells at the early exponential and latestationary phases (days

3, 9, and 12) as at the late exponential/early stationaryphase (Fig. 3). This

dramatic loss of GalNAc and surprising increase in GlcNAc(as seen with

FITC-WGA) may indicate the metacyclogenic stage of theparasite, as seen

in in vitro cultures. As the FITC-WGA and FITC-SBA gavesuch interesting results in the

foregoing study, I have been using these lectins with otherstrains and Table 3 Percentage of Cells Labeled byFITC-Lectins for All Strains Tested Under the Same GrowthConditions in Semidefined Media and Harvested at theStationary Phase Species and FITC-Lectin LRC number PNAConA SBA WGA RCA L. major L306 70.9 79.7 19.4 39.7 85.3L464 39.0 84.2 46.2 39.8 95.0 L23 69.7 90.5 40.7 21.7

90.9 L38 45.6 86.6 41.6 39.6 81.2 "P" 60.0 56.7 52.830.0 82.4 L505 27.6 75.3 36.4 38.0 84.7 L. donovaniL133 0.11 99.0 1.02 60.5 91.2 L. aethiopica L1470.64 91.3 0.38 75.4 96.4 L. amazonensis L259 1.2 94.3 0.987.0 49.9 L. enriettii L327 0.0 56.5 0.1 40.0 67.5Source: From Refs. 42 and 58. 100 ~WGA 90 80l.lilll!JJ!ilB SBA ~ 70 C=:J PNA G) 60 > 50 ;:'iii 40 0 30 a.. 20 10 0 3 6 9 12 Day Figure 3Fluorescent lectin-labeling of L. major (LRC-L544)promastigotes during the growth cycle. (From Ref. 36.)

species of Leishmania grown in different media. Thepercentage of L. don

ovani (LRC-Ll33) promastigotes, grown in biphasic medium(NNN), that

are labeled with FITC-WGA increased with time until thelate stationary

phase of growth (about day 9), when there was a rapiddecline in the

number of cells labeled. When the same cells were culturedin a semidefined

medium (SDM), only a steady increase of binding, duringgrowth, was

observed. The FITC-SBA did not label any L. donovani cells.When L.

tropica (LRC-L36) promastigotes were similarly labeled, thedramatic re

duction (that was also seen in L. major [36]) of thepercentage of cells that

lose the ability to bind to FITC-SBA during the stationaryphase was also

observed, but only in the NNN medium. In the SDM medium,the promas

tigotes of this strain were poorly labeled with FITC-SBAand seemed to

lose any GalNAc-binding sites after the fifth day inculture. These results

indicate that the parasites adapt rapidly to thesurrounding medium and

changes occur in the surface carbohydrate topography. Thesechanges may

reflect the various sandfly diets that the parasites areexposed to in nature. Table 3 summarizes the results fromthe F ACS studies and shows the

percentage of binding to each FITC-lectin that was used.All cells were

grown in semidefined medium and harvested at the stationaryphase of

growth. Only those strains that were examined with thepanel of five lectins

are tabulated.

B. Horseradish Peroxidase and Ferritin

Several authors have used horseradish peroxidase (HRPO) anddiamino

benzidine (DAB) or ferritin and radioactive 3 H-labeledlectins to map the

surface membranes of Leishmania [10,27,43]. These studiesused electron

microscopy to visualize the actual location of thelectin-binding sites on

the surface of the parasites. When using ConA, anasymmetric coat of

a-o-glucopyranosyl, a-o-mannopyranosyl, or{j-o-fructopyranosyl was dis

tributed over the surface of L. braziliensis guyanensis toa mean thickness

of 50 nm [27]. In a thorough investigation of the surfaceof L. donovani

(clone 1-S, Cl 2 _ 0 ), HRPO-ConA, HRPO-SBA, and HRPO-WGAwere

used (as well as 3 H-labeled lectins) to map surfacecarbohydrates and follow

the kinetics of binding [10]. Dense ConA-HRPO-DAB reactionproduct

was restricted to the pellicular and flagellar outerlamina. Cells treated with

SBA-HRPO or DAB had no dense-staining products, but whenused in

combination (SBA-HRPO-DAB}, large amounts of the lectinreaction

product were noted on portions of the surface membranecovering the

subpellicular microtubules. Similar results were obtainedwith WGA

HRPO-DAB, with the additional observation that the lectindeposits were

also found on the flagellar membrane, within the flagellarreservoir, and 208 the membrane lining this cavity. Theamount of cell-bound [ 3 H] ConA was determined from meansaturation-binding kinetics, which was calculated to be2.78-4.86 x 104 /Lmol/10 8 which probably represents theminimal number of a-o-mannose ligands stericallyavailable for binding with the lectin and does not reflectthe total number of moieties present on the surface ofthe promastigote [10]. In another study of L. donovani,WGA-HRPO and PNA-HRPO were used to study stage-specificconversion of amastigote to promastigote and vice versa[43]. In this study, in which agglutination, FITC-lectins,and HRPO-lectins, all were applied to the two stages, theauthors showed that HRPO-PNA bound to the promastigote,but not to the amastigote. Conversely, HRPO-WGA boundhomogeneously only to the amastigote and not to the maturepromastigote. During stage conversion, from amastigote topromastigote, the HRPO-PNA was first found inside theflagellar pocket of the promastigote and, later, over theentire surface of the promastigote. This presumed loss ofPNA-binding sites in the infectious metacyclic form hasbeen the subject of much debate in several papers[20,46,47], but the significance of WGA-binding sites hasbeen mostly ignored [36]. C. Enzyme-Linked Lectin AssayThe lectin receptors of formalin-fixed promastigotes wereprobed using enzyme-linked lectins. The parasites of L.

d. donovani, L. d. chagasi and L. m. amazonensis werefixed in formalin and attached with poly-L-lysine tomicrotiter plates. The parasites were then incubated withenzyme-linked ConA, RCA, WGA, PNA, and SBA. Only theparasites of the L. d. chagasi strain reacted with SBA,but all three strains reacted with the other lectins.Trypsinization of the cells did not remove the lectinreceptors [48] as had been previously shown [49]. IV.INFECTIVITY OF LEISHMANIA AND LECTINS The fact thatlectins agglutinated promastigotes differently, dependingon the phase of growth, was described over 10 years ago,and these differences were correlated with the infectivityof L. donovani promastigotes for golden hamsters [19]. A.Leishmania donovani and Ricinus communis Lectin The threelectins used for this study were ConA, RCA-I, and RCA-II,and the strain of L. donovani was the Sudanese 3S strain.The results showed that 10-day cultured promastigoteswere less agglutinated than 3-day cultured cells whenincubated with low concentrations (25 /Lg/ml) of RCA-I

and ConA. Of these 10-day promastigotes, about 150Jo couldestablish

themselves in macrophages, whereas fewer than 2% of 3-daycultures would

infect the mammalian cells. The authors concluded that, asRCA-11

mediated agglutination did not change over time,a-D-galactose was con

stant, but as RCA-I binding did, GalNAc was the variantcarbohydrate on

the surface.

B. Leishmania major and Peanut Lectin

Another group, using L. major (Friedlin strain NIH, Clone1A), and the

lectins ConA, RCA-II, SBA, WGA, UEA-1, and PNA atconcentrations

from 0.37 to 100 p.g/ml, showed with this strain thatexponentialand

stationary-phase promastigotes varied only with PNA, andless so with

RCA-II [20,46]. Here, about 10% of the 3-day promastigoteswere infective

to mouse peritoneal cells and to 50% of the 5-day-culturedcells.

C. Leishmania donovani and Peanut Agglutinin

As PNA agglutination was thought to be a suitable markerfor the infectiv

ity (metacyclogenic) of cells, other groups tried torepeat the work [46] with

different strains. An L. donovani strain (MHOM/ET /67/HU3) was grown

in a specialized medium (modified Gracie's medium) fromamastigotes de

rived from hamster spleen [47]. Three lectins (ConA, WGA,and PNA)

were used to test the changes in agglutination betweenexponentialand

stationary-phase promastigotes. The WGA failed toagglutinate any cells,

ConA binding was the same for both stages, but only about20% of S-phase

cells agglutinated with PNA (125 p.g/ml) against almost100% of theE

phase cells. The authors concluded that the change insurface galactose, as

promastigotes matured toward metacyclogenesis, wasprobably a common

phenomenon of the Leishmania. A method for purifyingpopulations of

PNApromastigotes has been published in which sucrosegradients were

used [50].

D. Leishmania enriettii and Other Lectins

A different approach to the problem of variation on thesurface of the

promastigote during maturation was that of comparing aninfective and a

noninfective strain of the same species. Two stocks ofL. enriettii, with differing pathogenicity to guinea pigs,

were studied according to their lectin-mediatedagglutination with the fol

lowing lectins: ConA, RCA-II, EUE, SOH, PNA, UEA-1,UEA-11, LO

TUS, and LAA [51]. The FITC-UEA-1, FITC-PNA, and FITC-LOTUS

complex, were also used to ascertain differences betweenthe two stocks. The concentrations of lectins in thisstudy were high compared with other studies (seeforegoing), as 1000 J.Lglml was an average for most tests.The EVE and UEA-1 were negative for both stocks. At thesame concentration ConA, PNA, LAA, and UEA-11 werenegative for the pathogenic stock and only LOTUS wasnegative for the nonpathogenic stock. The FITCLOTUS complexlabeled only the pathogenic stock, whereas FITC-UEA-1labeled neither stock. The main conclusion that can bederived from this study is that the two stocks differquite radically in their surface carbohydrates. Theapparent lack of ConA receptors (indicating loss ofmannose residues) is quite different from other studies,as the parasites of both stocks were harvested at S-phaseand, for most Leishmania strains, no difference has beenreported. Although LOTUS lectin strongly agglutinated thepathogenic stock (fucose receptors), UEA-1 failed toagglutinate either stock. Similarly, UEA-11 and LAA[(GlcNAc) 2 and Fuc,Gal] agglutinated only thenonpathogenic stock, indicating receptors for the LOTUSlectin and N,N-diacetylchitobiose as membrane componentsof the New World nonpathogenic L. enriettii stock. E.Leishmania braziliensis panamensis and Concanavalin A andLentil Lectins Another New World species of human originthat has been studied for differences in lectin binding,during in vitro growth, was L. braziliensis panamensis(strains MHOM/PA/82/WR470 and MHOM/PA/83/WR539) [52]. Thepanel of FITC-lectins tested included PNA, WGA, SBA, UEA-1,DBA, ConA, and LCA. Only the two mannose-glucose-specificlectins (ConA and LCA) bound the promastigotes, and LCA

bound to only the stationary phase parasites. Otherlectins were used to study the glycopeptides of thisparasite and this will be discussed later. F. Leishmaniamexicana amazonensis and Other Lectins One of the morecomprehensive studies of the interaction between lectinsand a New World strain of Leishmania entailed the use of28 highly purified lectins, and a binding assay with 1251-labeled lectins [49]. The strain used was L. mexicanaamazonensis (Josefa strain), originally isolated from ahuman case of cutaneous leishmaniasis, and bothamastigotes (with and without membranes) and promastigotes(infective and noninfective) were tested. NineGalNAc-binding [JCA, BS-1( =GS-1), DBA, SBA, HPA, MPA,LBA, PHA, and WIF], four Gal-binding (AXP, PNA, RCA-I, andRCA-II), three GlcNAc-binding [AAP, BS-11 (=GS-11), WGA],two Man/ Ole-binding (ConA and LCA), and a sialic acid(LIP or limulin)-binding lectin, all were positive for thepromastigotes. Except for LBA, RCA-II,

Lectin-Leishmania Interactions 211

and LPA (which were not tested) they would also agglutinatewith the

amastigotes. The specific results indicate that LBA, WIF,and WGA react

only with infective forms, whereas PNA and AXP were moreselective for

infective forms (ten times more lectin required toagglutinate the noninfec

tive forms). Hence, for this parasite, GalNAc, GlcNAc, andGal residues

are all increased in the infective promastigote form. Thediscrimination of the binding capacity of amastigotes frompro

mastigotes was most noticeable where BS-1 ( = GS-1), DBA,PHA, SBA,

and WIF are highly specific for amastigotes and MPA forpromastigotes.

Lectins that did not react to either stage included LOTUS,UEA-1, SOJ,

BPA, VVA, STA, PWM, UEA-11, andGEC. The binding and

kinetics of 125 1-labeled lectins (WIF, WGA, and PNA)

were also studied by the same group [49]. Infective andnoninfective pro

mastigotes were easily separated by this method. With thesethree lectins

the number of binding sites per infective promastigotecould also be calcu

lated. The ratio of binding sites for WGA/WFA/PNA was30:7.9:3.1,

indicating that for this strain of L. m. amazonensis,GlcNAc was more

abundant than GalNAc and Gal.

G. Leishmania major and Fluorescein lsothiocyanateLabeled-Lectins

The problem of what constitutes an infective(metacyclogenic) promastigote

has also been studied by following the growth of theparasites in culture

and sequentially labeling with FITC-lectins. The populationof labeled par

asites was then analyzed by flow cytometry (F ACS) andsamples of the

populations injected into hamsters at each phase of thegrowth cycle [36].

The FITC-lectins used were PNA, WGA, and SBA. The parasitewas a

new human isolate of L. major (strain MHOM/IL/87/YD;LRC-L544). Promastigotes of the strain of L. majorLRC-L544, grown in SDM,

showed a diverse pattern of labeling when incubated withFITC-lectins

during the growth cycle (see Fig. 3). Most ofthe cells(82-90%) were readily

labeled with FITC-PNA throughout the entire growth period.When the

cells were incubated with FITC-WGA there was an increase inthe percent

age of cells labeled, from 27o/o on day 3 to 61 OJo on day12. When the

promastigotes were labeled with FITC-SBA, a reduction ofbinding was

reported, from 75% on day 3 to 40% on day 6. This trend wasreversed by

day 9 with 80% of the cells being labeled (Fig. 4). Whenthe parasites

with the different carbohydrate configurations, wereinjected into golden

hamsters, there was no appreciable differences in thelesions caused, regard

less of the age of the culture (Table 4). The constancy ofFITC-PNA

binding in a virulent parasite population was surprisingwhen compared 212 Jacobson Q) PNA c WGA SBA c 0..c u .......... 3 days (/) Q) u 0 loQ) ..c E:;) c Q) > +0 9days Q) a:: Relative intensity offluorescence Figure 4 Flow-cell cytometric histograms offluorescent lectin labeling during the growth of L. majorLRC-L544 promastigotes. (From Ref. 36.) with theappearance of PNApromastigotes in other strains of the samespecies [38]. The presence ofWGA receptors (GlcNAc), thatincreased over time and the decrease and reappearance ofSBA receptors (GalNAc) during a long growth cycle,indicated a change in the surface carbohydrateconfiguration that overlapped the amastigote surfacecarbohydrates described for virulent L. donovani [43].Table 4 Infectivity to Hamsters of L. major (StrainLRC-L544) Promastigotes from Different Days of CultureDays after Days of growth infection 3 6 9 21 1/3 1130/3 42 3/3 3/3 2/3 63 3/3 3/3 3/3 Source: Ref. 18.

V. LEISHMANIAL GL YCOCONJ UGATES AND LECTINS

One of the most interesting developments in the study ofleishmaniasis

has been the exploration of the leishmania!glycoconjugates. The contact

between the parasite and the host macrophage or the sandflygut microvilli

is the cell surface. Within this surface membrane isanchored a unique

lipophosphoglycan, which is released into the surroundingmedium. This

material, it has been suggested, is multifunctional [7]and has been used as

the basis for serotyping (taxonomy) [13], cell biology,and vaccination stud

ies. Lectins have been particularly useful in defining theoligosaccharides

that are the distal portion of the molecule.

A. Glycoconjugates and Surface Membranes

The presence of carbohydrate residues in the pellicularmembrane (PM)

was demonstrated by isolating the membrane from L. donovani(strain

1-S, clone 2-D}, exponential-phase promastigotes [53]. Themembranes

were solubilized in SDS, run on sodium dodecylsulfate-polyacrylamide gel

electrophoresis, (SDS-PAGE), and probed with FITC-lectins.The FITC

lectins used were ConA, DBA, RCA-I and II, SBA, UEA-1, andWGA. All

the lectins "stained" the PM bands, and it was suggestedthat PM carbohy

drate ligands were side chains on membrane glycopeptides orglycoproteins.

When lectin-ferritin conjugates were used, all the lectinsbound only to

the external lamina of the PM and, therefore, thecarbohydrates' spacial

orientation was external to the membrane plane, and therewas chemical

asymmetry of the PM relative to glycosylation [53].Another method of comparing species and strains for theirmembrane

glycoconjugate components is that of lectin blotting. Thismethod has been

used both on purified membranes [54] and extracted wholepromastigotes

[52,55]. Purified membranes of several species were probedwith either

125 1-ConA or by triple sandwich Western blots for LCA,SBA, PNA, and

RCA-II [54]. The strains tested were L. major; L. b.braziliensis, and L. b.

panamensis,· L. m. mexicana and L. m. amazonensis,· L.tropica,· L. dono

vani infantum and L. enriettii. The results showed thatthe triple-sandwich

technique for ConA gave nonspecific binding; therefore, thedirect method

was used for this lectin. Only the two species from the L.mexicana complex

were essentially similar in the overall patterns.Concanavalin A and LCA

bound to similar glycoproteins, with multiple componentswith relative

molecular masses (Mr) ranging from 27,000 to 200,000. OnlyL. b. brazil

iensis and L. enriettii were exceptional with single

components of Mr 52,000

and 200,000, respectively. High Mr doublet or triplet(160,000, 175,000, and

185,000) polypeptides are present in the other stocks.Leishmania tropica

(LRC-L36) was the only strain that did not react with thegalactose family of lectins (RCA, PNA, or SBA). The PNAbound to only three strains: L. b. panamensis, L.enriettii, and L. major (LRC-L137). The other strain of L.major (LRC-L251), which had been in continuous culture for13 months (and, therefore, nonpathogenic), was weaklypositive for PNA, but negative when freshly isolatedpromastigotes were cultured from mouse lesions.Lectin-hlotting with PNA selectively binds to 28,000,37,000, and 48,000M. membrane components of both thenon-pathogenic and the weakly pathogenic strains of L.major [54]. B. Glycoconjugates of Extracted PromastigotesA different approach to the study of the constituentcarbohydrates of the leishmanial glycoconjugates was usedby extracting late exponential-phase parasites byfreeze-thawing, separating on SDS-PAGE, and running thecomponents in Laemmli gels [55]. The lectins wereradioactively labeled, using the lodogen® method, and usedto stain the gels. Fourteen different strains ofLeishmania were probed with 14 lectins. The SBA, SNA, andNarcissus pseudonarcissus (daffodil; specific for terminala-mannose) did not bind to any component. The LYE, GAN,Sus scrofa (porcine lung lectin; PLL; 13-Gal), andColchicum autumnale (autumn crocus; Gal/3,4Glc > GalNAc >Gal) bound to the 11-kDa band in all strains, as did mostof the lectins. The PNA bound to only the 11-kDa band inL. b. panamensis and L. m. amazonensis. The GlcNAc-specificlectins DAS (the same four bands for all species 11, 19,50, and 86 kDa) and WGA (multiple binding to differentbands according to species) showed a wide range ofglycoconjugate specificities. A 62-kDa band wasconsistently labeled (8 of 14 species) with theMan-Gle-specific lectin ConA, but not by PEA lectin. Otherlectins used included ST A, which bound to only the twostrains tested from the L. donovani complex, and GS-1,which bound to the 24-kDa band of L. b. braziliensis, a53-kDa band of L. m. mexicana, and a 63-kDa band of L. d.donovani. The conclusion drawn from this study, whichsometimes agreed [14] and sometimes contrasted [13,36]with previous studies, was that it is difficult toextrapolate from lectin-mediated agglutination results theidentity of the carbohydrate moieties of individual

glycoproteins [55]. In another study, extracted L. b.panamensis exponential-phase and stationary-phaseparasites were blotted and probed with theperoxidaselabeled lectins DBA, GS-1, GS-11, ConA, PNA,WGA, MPA, RCA-I, SBA, and UEA-1 [52]. Only the lectinsPNA, WGA, DBA, and MPA bound to the Western blots; PNAbound to two glycopeptides (M. 59 and 61 kDa) ofstationary-phase promastigotes, but not to blots ofexponential (E)-phase cells. These unique glycopeptidebands of the S-phase were also 215

intensely stained by the other lectins, but so were otherbands from E-phase

promastigotes. Especially noticeable were three high M,bands (152, 165,

170 kDa) that were stained with WGA and were unique toE-phase promas

tigotes. There were some differences between these results[52] and the

other reports [54,55], but the methods and individualisolates were not

identical, so it is difficult to make a direct comparison.

C. Released Glycoconjugates

We have already seen how some leishmanial investigatorsfelt that the ab

sence of receptors for PNA (i.e., PNAcells), wasindicative of infectivity

[46], whereas others showed that if macrophages had surfacereceptors for

galactose, then this would be the most likely candidate foran immunodomi

nant carbohydrate [56]. In this latter study, only RCA-Iand RCA-II could

precipitate the released glycoconjugate (EF), whereas ConA,WGA, DBA,

LOTUS, and SBA, all failed to precipitate EF from the L.major strain

LRC-L137. The theory suggested that theleishmanial-released glycoconju

gate was, therefore, a macrophage-conditioning agent,facilitating the rapid

uptake of cells, and a lymphocyte inhibitor. RCA-II waslater used to

purify, by affinity chromatography, the releasedglycoconjugate in an at

tempt to ascertain the saccharide and amino sugar contentof the material

from L. major and L. donovani [57]. The absence of GlcNAcand the

presence of xylose as reported [57] differ somewhat fromthe currently

accepted chemical structure of the glycoconjugate of thesetwo parasites

[21,22]. In cooperation with Dr. L. F. Schnur, I havebeen studying the under

lying fundamental differences in released glycoconjugatesof Leishmania

of the B serotype complex. This group of organisms includesL. donovani

(LRC-L133), L. aethiopica (LRC-L147), L. amazonensis(LRC-L259), and

the nonpathogenic L. enriettii (LRC-L327). Each of thesefour species have

different subserotypes, so it was of interest to determineif their differences

were detectable using FITC-lectins [58].{3-D-Galactose(1-3)DGalNAc is almost undetectable on thesurface of

stationary-phase promastigotes of serotype B strains whenlabeled with

FITC-PNA and FITC-SBA, so these carbohydrates could notaccount for

the differences. a-D-Mannose and a-D-glucose (FITC-ConA),(D-GlcNAc) 2

(FITC-WGA), and {3-D-galactose (FITC-RCA-II), are allpresent, but only

50"7o of L. enriettii cells could be labeled with ConA, andonly 50"7o of L.

amazonensis were labeled with RCA. Thus, there was adifference in the

mannose/galactose ratio for these two strains. The FITC-WGAlabeling

showed the greatest diversity among the strains. Over85"7o of L. amazonen

sis cells were labeled [with 50"7o inhibition by (GlcNAc) 2], whereas only 216 Jacobson 350Jo of L. enriettii cellswere labeled [only 10% were partially inhibited by(GlcNAc) 2 ] with the same FITC-WGA; 60% of L. donovanicells were labeled [with only 25% inhibited by (GlcNAc) 2], whereas 75% L. aethiopica promastigotes bound to thislectin and there was virtually no inhibition by the(GlcNAc) 2 (Fig. 5). An examination of the histograms fromthe flow cytometry showed that the numbers of cells andthe relative intensities of fluorescence were verydifferent (Fig. 6). We believe these differences are thereason for the serotypical differences found among thesestrains [58]. VI. CYTOTOXIC LECTINS AND LEISHMANIA Thecytotoxic effect of lectins on leishmanial parasites wasfirst reported in taxonomic studies, in which it wasreported that cells agglutinated by RCA-I were notreleased by the addition of 0.5 M lactose nor was thepronounced toxic effect reversed [13]. Parasites thathave been exposed to mutagens and then grown selectively inculture media with different concentrations of RCA-I,resulted in the production of ricin agglutinin-resistantclones [59]. The mutagen used was N-methylnitroso-N'-nitroguanide, at a concentration of 3.5 ILg/ml for 3.5hr on L. donovani promastigotes. The parasites weresubsequently exposed to 100 ILg/ml of ricin on 2%agar-supplemented Dulbecco's modified Eagle medium (dOME)plates. Clones were selected on the basis of resistance toRCA-I agglutination, and one was subsequently furthercharacterized (the R2D2 clones). The clones retained their

resistance to the ricin lectin, even when grown in normalcondition, but became much more sensitive toagglutination with ConA. The R2D2 clones and wild-type L.donovani promastigotes were exposed to ricin-gold labelingand only the wildtype cells were labeled. These clones werealso incorporated in greater numbers by macrophages thanwere the wild-type promastigotes, as shown by [ 3H]uracillabeling. The conclusions drawn from these studiesindicate the R2D2 clones did not possess theglycoconjugate lipophosphoglycan, and that they would beuseful in future investigations in dissecting themolecular structure of the parasite. VII. LECTINS AND THESAUROLEISHMANIA In view of the new taxonomic status of theLeishmania, such as parasites of reptiles (they areactually a different genus, Sauroleishmania), it was feltnecessary to keep them separated from the mammalianLeishmania [1]. Sauroleishmania tarentolae senagalensis(G.10) and S. adleri (LRCL123) promastigotes were exposedto 23 lectins of diverse specificities [16].

lectin-leishmania Interactions

Q) 80

>

VI

0

0.

~ 0 60 40 20

Q) 80

>

VI

0

0.

~ 0 60 40 20

Q) 80

>

!::

VI

0

0.

~ 0 60 40 20 0 L.donovani L.aetlliopica LamazonensisL.enriellii Ldonovani L.aethiopica l.amazonensisL.enriettii l.donovani Laethiopica l.amazonensisL.enriettii 217 EJ ConA • Con +man E'JRCA • RCA +gal('JOCA • WGA+ GluNac

Figure 5 Fluorescent-lectin labeling of four B serotypeLeishmania and the effect

of inhibiting carbohydrates. Cross-hatched, labeled byFITC-lectin; black, labeled

in presence of carbohydrate. Cl> c c 0 ..c u ' C/)Cl> u 0 .... Cl> .0 E ::J c Cl> > .B Cl> 0::L. donovani L.aelhiopica L.amazonensis L.enrie11i Con ARCA WGA Relative intensity of fluorescence Figure 6Flow-cell cytometric histograms of FITC-lectin labeling offour different species of Leishmania. The four speciesare all B serotype. There were no reactions to fucoseorN-acetylchitobiose-binding lectins and little reactivityto glucose or mannose-specific lectins, except for ConA.Four lectins, SRA (Gal > > Fuc), CLN (GalNAc), and RCA-Iand RCAII, gave strong agglutination reactions with thesetwo stocks. The authors concluded that the lectin profileof the Sauroleishmania could not be superimposed on anymammalian strain [16]. A strain of S. agamae (LRC-L121)was tested for the ability of its promastigotes toagglutinate with UEA-11; a weak reaction was reportedthat was inhibited by 80 mM of lactose [34]. Thecarbohydrate moieties of S. adleri glycoconjugates havebeen partially characterized by ConA affinitychromatography [60]. The results of this studydemonstrated that the a-o-manno-pyranosyl andgalactopyranosyl units were associated with the samepolymer, and a galactomannan remained specifically attachedto the ConA. 219

VIII. CONCLUSION

Lectins have been used widely in the study of theleishmaniases both for

their surface carbohydrate moieties and theirglycoconjugates. The lectins

have helped dissect the molecular structures of both thepromastigote and

the amastigote. They have been used as taxonomic tools,infectivity and

virulence markers, and for the purification of leishmanialproducts. The

knowledge gained from the labeled lectins has greatlyincreased the under

standing of carbohydrate configurations on the surfacemembranes of these

ubiquitous parasites. The oligosaccharides on and withinthe surface mem

brane of the parasites are the key to their survival inthe hostile environment

of the macrophage phagolysosome or the alimentary tract ofthe sandfly

vector. If the disease leishmaniasis and the epidemics itcauses are ever to be

controlled, it may be through the basic understanding ofthe carbohydrate

profiles of the glycoproteins and other glycoconjugatesthat have been char

acterized with the aid of lectins.

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9

Microbial Lectins for the Investigation

of Glycoconjugates

K. L. SHAKHANINA and N. L. KALIN IN Gamaleya Institute of

Epidemiology and Microbiology, Russian Academy of MedicalSciences,

Moscow, Russia

V. M. LAKHTIN Institute for Applied Science of MoscowUniversity

and Institute of Food Substances, Russian Academy ofMedical Sciences,

Moscow, Russia

1. INTRODUCTION

This review contains data on interaction of lectins ofviruses, rickettsias,

bacteria, protozoans, microscopic fungi, and yeasts withsoluble and recep

tor glycoconjugates (glycoproteins, glycolipids,lipopolysaccharides, and

other biopolymers) containing polysaccharides ofvertebrates, inverte

brates, plants, and microorganisms. The summary presentedindicates not

only carbohydrate (monoand disaccharide) andoligosaccharide extended

specificities of lectins from different microorganisms,but also defines their

specificities toward glycoconjugate polysaccharidemolecules. The sum

mary may be used as a basis for purification of receptorsand other glyco

conjugates and polysaccharides with the help ofimmobilized microbial

lectins. In recent years more and more attention has beenpaid to carbohy

drate-binding proteins, especially lectins. This isclearly evident as seen

from numerous reviews and books [1-11]. Multipleconferences, symposia,

seminars, and schools have been devoted to the study ofglycoconjugates

and lectins. Rapid growth in the numbers of firmsproducing complex

glycans, neoglycoconjugates, lectins, glycosidases,carbohydrate-binding

toxins, monoclonal antibodies to carbohydrate-containingtargets, and

publication of new scientific journals (e.g., Trends inGlycoscience and

Glycotechnology) are additional evidence for theimportance of studying

glycoconjugates and their interactions with lectins. Infact, glycoconjugate

recognition is the basis of several key me~hanisms ofbiological recognition

of living organisms. 299 The least studied lectins arestill those of microbial origin that interact selectivelywith glycoconjugates of host organisms (initial infectionprocesses in humans, farm animals, plants, and marine foodorganisms). Aspects of bacteria-mediated fixation ofnitrogen in tubercles of plants, the obtaining ofmicrobial insecticides, the phenomena of microparasitism,and selective coaggregation of microorganisms, allinvolve protein-glycoconjugate interactions [4, 12-17]. Inmany interactions between microbiallectins and cellsurfaces, there is insufficient information available aboutthe structures of receptor glycoconjugates and the spectrumof possible glycoconjugate targets. Study of theinteraction of purified microbiallectins with a set ofmodel glycoconjugates (glycoproteins, glycolipids,polysaccharides) with the known structures of glycanshelps define the specificities of microbial lectins.Therefore, the main task of the present review is tooutline the data on new preparations of microbial lectinsthat have not been considered in other reviews [12,18-22].II. INTERACTION OF MICROBIAL LECTINS WITHCARBOHYDRATE-CONTAINING TARGETS Agglutinins (mainlylectins) have been investigated from about 150 microbialsources and isolated from only a few species. However,lectins from only 100 microorganism species have beencharacterized for monosaccharide specificities. There islittle information about oligosaccharide specificities ofmicrobial lectins or of their specificities for wholemolecules of glycoconjugates and polysaccharides. The main

sources of microbiallectins are from viruses (defined asa microorganism for purposes of this book), bacteria,protozoans, yeasts, and fungi, including mushrooms. Thelectins that belong to each of these groups ofmicroorganisms will be considered. Ill. VIRAL LECTINSTable 1 shows some results on the interaction ofvirallectins with receptors and isolated glycoconjugates(natural or synthetic). Many viral proteins showhemagglutinating activities [23], although it is onlyrecently that hemagglutinins of viral origin have begun tobe considered as lectins [23]. Carbohydrate-containingtargets for viral lectins, as a rule, includelipopolysaccharides, gangliosides, and glycoproteins (seeTable 1). The most thorough investigation was carried outon the specificities of the hemagglutinins of influenzaviruses for mammalian glycoproteins and gangliosides[34-39]. All hemagglutinins of influenza virus types A, B,and C exhibit the properties of sialospecific lectins,interacting with terminal residues of N-acetylneuraminicor N-glycolylneuraminic acid in glycans. 301

Table 1 Interaction of Viral Lectins with Glycoconjugatesand Polysaccharides Targets containing

Source of lectins carbohydrates Ref.

Bacterial viruses Bacteriophage 013 hemRa, Rb 1 , Rb 2-type LPS from mutant 24 agglutinin strains ofSalmonella sp., Escherichia coli. Bacteriophage <!>X174Glc-a-1 ,2-Gal-a-1 ,3-Glc-containing 25 hemagglutininLPS of Salmonella

Coronaviridae Sheep encephalomyelitis9-0-Ac-Neu5Ac-containing protein 26 virus and bovinecorona of surface structure of chicken virus hemagglutininerythrocytes and human sialidasetreated erythrocytes

Hepadnaviridae Hepatitis B virus S proNeuAc,NeuAc-Lac-containing sur27 tein face structures of Verocells

Herpesviridae Herpes simplex virus type Sensitive toheparin surface structure 28 1 and pseudorabies virus ofrabbit kidney cell lines RK-13 GPll-B, GP111-C, GPV

Lentiviridae Human immunodeficienAntigen CD4 andtransmembrane 14,29 cy virus type 1 gp120 gp41 ofTlymphocytes

Reoviridae Reovirus type 3 0 proGlycoprotein A MN of

human erythro30 tein 1 cytes Orbivirus of blue linguaeNeu-Ac-a2,6, NeuG-a2,6-containing 31 of sheephemagglutinin (GP); glycophorin, mucin of bovinesubmandibular gland

Rhabdoviridae Rabies virus hemaggluGangliosides GTlb,GQ1b, GDlb 32 tinin Vesicular stomatitis viralGangliosides GM 3 of goose erythro33 hemagglutinin cytes

Orthomyxoviridae Influenza virus type A NeuAca2,3(6)NeuG-a2,3(6)34,36,37 containing gangliosides and GPNeu-Ac-containing a 2 -macroglobulins Influenza virus typeC Neu5Ac-a-containing neopolysac38 hemagglutinin charide9-0-Ac-NeuAc-containing protein of 39 surface structureof chicken erythrocytes However, the hemagglutinin ofinfluenza virus type A predominantly recognizes4-0-acetylated groups of N-acetylneuraminic acid.Influenza virus type C hemagglutinin shows a high affinityfor 9-0-acetylated derivatives of sialic acid [37-39].Moreover, influenza virus lectins are capable ofrecognizing microdomains and clusters from the sialic acidresidues within the elongated glycans, including sets ofsialic residues. Some viral lectins are specific forpolysialogangliosides (e.g., rabies virus hemagglutinin),and other virallectins for mannosialogangliosides (such asthe vesicular stomatitis virus hemagglutinin) [32,33].Specificities of virallectins for lipopolysaccharide andneopolysaccharides with known structures have also beenstudied [24,25]. Because of the density of glycanclusters, their lengths and glycoconjugate structures, andcomposition variations, it is possible to detect glycanswith maximal affinity for lectins, as has beendemonstrated for hemagglutinins of bacteriophage G13 andinfluenza viruses [24]. Chapter 2 of this book provides adetailed examination of the interaction between lectins,viruses, and viral-infected cells. Ill. BACTERIAL LECTINSSignificantly more papers have been published onspecificities of bacterial lectins [15-22,24,41-44]. Thismay be because the researchers began to considercarbohydrate-binding toxins of bacterial origin as lectins[41 ,53 ,54]. Among the sources of bacterial lectins,several investigators report nearly 100 strains from morethan 70 species of organisms. However, most of the lectinshave not been purified, or the purified lectins have notbeen exhaustively investigated from the viewpoint oftheir specificities for glycoconjugates [18]. Table 2 givesdata on the known specificities of bacterial lectins. Ascan be seen from Table 2, bacterial lectins representpreparations with different biological activities andcellular distribution. Adhesins may be found on microbial

surfaces containing fimbria! and nonfimbrial appendages.Some are extracellular toxins, and some are enzymes (seeChapter 1 for a discussion of the definition of a lectin).All the microbiallectins, as a rule, show hemagglutinatingproperties. Toxic hemagglutinins usually includeRNA-N-glycosidase or ADP-ribosyltransferase activities[41], although frequently proteolytic activity is alsodetectable in microbial hemagglutinins [18,139]. Someglycosidases and glucose oxidases have, apart from theircatalytic centers, lectinlike domains [44, 117, 179].From the examples of coliform bacteria, pseudomonads, andcholera vibrios one can observe that each of themicroorganisms synthesize a set of lectins with variousspecificities for glycoconjugate targets (see Table 2).Lectin sources Actinomyces naeslundii WVU45 fimbriae A.viscosus T14V fimbriae Aeromonas caviae, A. hydrophila, A.veroni, A. sobria hemagglutinins pilins/ adhesinsAgrobacterium tumefaciens lectins Azospirillum brasiliensehemagglutinin Bacillus mesentericus 316M extracellularhemagglutinin Bacteroides jragilis adhesin (70 kDa) B.(Prevotella) loescheii adhesin Bordetella bronchisepticaadhesin B. pertussis pertussis toxin (B subunit oligomer)(11-22 kDa) adhesin (filamentous hemagglutinin)Bradyrhizobium japonicum Campylobacter jejuniextracellular enterotoxin Carbohydrate-containing targetsGal-~1 ,3-GalNAc/GalNAc-~1 ,3-Gal-containing glycolipidsGal-~1 ,3-GalNAc/GalNAc-~1; 3-Gal-containingpolysaccharides Sensitive to mannose of animalerythrocytes and lines of mammalian cells a-L-Fuc-BSA;fucan; chondroitin-sulfate; pectin of citrusa-L-Fuc-containing substances of erythrocytes NeuG 1;NeuAc-containing substances of rabbit erythrocytes a-GleN;a-GalN-Sepharose; polymers of the bacterium Enterococcushirae; epithelial intestinal cell line of human Proandeukaryotic cells NeuAc-Lac-Gp-containing sialogangliosidesof mycelia; mucin of bovine submandibular glandNeu5Ac-a2,6-containing GP; some asialo-GP; thermoprocessedfetuin; proteins of goose erythrocytes Substances ofcomplement-3/integrin aM ~ 2 ; CD 11 b + CD 18 ofmacrophages Lac-Sepharose; ~Gal; Lac-containing polymersof soybean cell line Gangliosides SRL ... Q =Ref. et42 ID 0. :r "' 42 45 46 47 48 49,50 51 5253-57 58,59 60,61 62,62a '-"'~ (continued) = '-"'~~ Table 2 Continued ~ Lectin sources Clostridiumbotulinum botulinum toxin type E C. botulinum botulinumtoxin C 2 C. difficile extracellular enterotoxin A C.spiroforme C. tetani tetanus toxin Eikenella corrodensEscherichia coli adhesin from type 1 (hemagglutinin 28kDa) fimbriae (or pili) Fimbrial type 1 of strain RDEC-1P-fimbriae (G adhesins) hemagglutinins of uropathogenic

strains S-fimbriae CS 1, CS 2, CS 3 of uropathogenic andother enterotoxigenic strains Fimbriae K88ab fromenterotoxigenic strains for piglet intestinal mucosaFimbriae K99 of enterotoxigenic strains Fimbriae F41 ofenterotoxigenic strain Nonfimbrial adhesin NFA-3 Membranetransporter of glucose Carbohydrate-containing targetsGangliosides and phospholipids Pig thyroglobulin; mucinof bovine submandibular gland; human erythrocytes treatedby sialidase Gal-ad, 3-Gal-,81,4-GlcNAc-containingsubstances of rabbit erythrocytes; bovine thyroglobulin;antigens Lex, LeY 1 of intestinal epithelium of humansHuman erythocytes Sialoganglioside-containing materialsGal-containing receptors Erythrocytes of guinea pig;Sensitive to mannose-containing substances of erythrocytesof guinea pig Man 5 GlcNAc 2 -peptide Gal-a1, 4Gal,8-containing globosides (globo-A and other) of humanepithelial cells NeuAca2, 3-Gal-containing substances ofmammalian cells gp 40-42 kDa of mucous epithelial cellsurface of piglet intestine Glycophorin A; glycopeptidesof pig intestine mucins; NeuG1-paragloboside andNeuG1-GM3 Glycophorins of human erythrocytes GlycophorinA NN erythrocytes of human with blood group N-antigenGlucose-containing ligands Ref. 63 64 65 70 71 9922,43,72,73 74-77 77-79 80 81-83 84-85 86-88 89 9091 w = \11 for human and pig Serotype 1 toxinSerotype 2 toxin 1 Serotype 2 toxin 2 Thermolabileenterotoxin of strains pathogenic for chickens Toxinfrom pig edemic disease strain TB-1 (pCG 5) Erwiniacarotovora subsp carotovora hemagglutinin E. rhapoviticihemagglutinin Eikenella corrodens surface hemagglutininLegionella pneumophila hemagglutinin Listeriamonocytogenes surface lectins Mycobacterium smegma tisextracellular lectin Mycoplasma salivarum surfacehemagglutinins M. hypopneumoniae hemagglutinins M.pneumoniae adhesins Neisseria gonorrhoeae nonfimbrialadhesin N. meningitidis S-fimbriae of strains causingmeningitis in newborns N. subflava nonfimbrial adhesin-1(27 kDa) GMt> GD 1 y, GD 1 b, GD2, GM2, GM3, GDI•• GD 1• and GT 1 b gangliosides Substances of pig blood groupA + H asialo-GP: mucin of bovine and thyroglobulin Gb 4,Gb 3 gangliosides Sensitive to mannose; rabbiterythrocytes Asialofetuin, Gal-{j1 ,4-GlcNAc-containingsubstances of human erythrocytes Binds to galactosamineof mammalian erythrocytes Adheres to carbohydrates ofmammalian erythrocytes treated with sialidase Ole, L-FucN-containing neoGPs Yeast mannan and mycobacterialarabinogalactan Sialic acid receptors of sheeperythrocytes; human B group blood erythrocyte Sialoreceptors of turkey erythrocytesNeuAc-a2,3-Gal-containing GP, laminin, fetuin,

choriogonadotropin of humans; Gal(3-S0 4 )-{jlcontainingsulfated glycolipids of adenocarcinoma! cell line WiDrAsialo GMt> GM 2 , containing glycolipidsNeuAc-a2,3-Gal-{j1 ,4-containing substances of bloodvessel endothelial cells NeuAc-a2,3-Gal-{j1,4-containinggangliosides of human erythocytes 95 96 97 98 99 100101 102 103 104 105,106 107 108 109 (continued) ~Q a'\ Table 2 Continued Lectin sources Porphyromonasgingiva/is (Bacteroides gingiva/is) adhesin(s) Pseudomonasaeruginosa lectins PA-l PA-Il Adhesins of strain ATCC27853 Exotoxin A P. fluorescens subsp cellulosaendoglucanase domain Pseudomonas syringae phaseolicolahemagglutinin P. solanacearum hemagglutinin Renibacteriumsalmoninarum hemagglutinins (57, 58 kDa) Salmonella dublintype 1 fimbria! hemagglutinins Shigella dysenteriae type 1dysenteric toxin Staphylococcus aureus Wood 46 adhesinsS. haemolyticus S. epidermidis, S. warneri hemagglutininsS. saprophyticus surface hemagglutininCarbohydrate-containing targets Peptide (12 amino acids)rich in histidine and GP from human salivaGalactose-containing receptors of erythrocytes; substancesof blood group A + B Mannoseand fucose-containing GP ofyeasts, plants, mammals NeuAc, GlcNAc-containingsubstances of human kidney and lung cellsp-Aminophenyl-{3-n-thioGalp; fetuin {3-1 , 4-glucan(cellulose) Sensitive to N-acetylgalactosamineFucose-containing substances of rabbit erythrocytesRabbit erythrocytes and salmon spermatozoaMannose-sensitive substances of erythrocytes Gal-ad,4-Gal-{3-BSA; galabiose-containing globosides (Gb 3 andothers) of HeLa cells Fucose-containing GP: fibronectinN-Acetylgalactosamine-sensitive substances of horseerythrocytes N-Acetylgalactosamine andN-Acetylglucosamine sensitive substances of human urinarytract epithelium and horse erythrocytes Ref. 111,112113 114 115 116 117 96 118 119 120,121 122 123123,124 IN Q "' Streptomyces murinus extracellularaggregation factor of cells S. sanguis surface adhesinS. cricetus and S. sobrinus (extracellular or surfacelectins) Vibrio cholerae thermolabile enterotoxin of its"B"subunits (11.6 kDa) Fimbriae (monomer 16 kDa) ofstrain 0 1 biotype El Tor Hemagglutinin (62kDa) ofnon-OL V2 strain cells (nonfimbrial origin)Hemagglutinin/metalloprotease from culture fluid ofstrain CA401 V. furnissii adhesins/ chemoreceptors ofmarine bacteria Ureaplasma urealyticum adhesinsXanthomonas campestris hemagglutinins uin glycopeptidesand bovine substances of glycophorin AMN 131,3(6)-Glucans of sarcoma cell line, Hela cells, E. coli,Bacillus subtilis, Micrococcus /uteus, Staphylococcus au

reus NeuAc-a2, 3-Gal-131, 3-GalNAc-sensitiveglycoconjugates of human saliva Glc-a 1 ,6-containinglinear glucans GM 1 , GM 2 , asialo-GM 1 ,neoglycolipids; (GM 1 ) oligopolylysine; substances ofmouse intestine epithelial cell nuclei (probably GP),glycoproteins with activity of A blood group from mucousmembrane of pig stomach Animal erythrocytes Fetuinandasialofetuin-sensitive substances of rabbit erythrocytesErythrocytes of the chicken breed "white leghorn" GlcNAc,Man, Glc-Sepharose; (GlcNAc) Glycophorin and dextransulfate-sensitive substances of human erythrocytesGlucosamine-containing substances of rabbit erythrocytes;xylose-, galactose-, N-acetylgalactosaminesensitivesubstances of rabbit erythrocytes GP, glycoprotein;NeuAc, N-acetylneuraminic acid; NeuG, N-glycolylneuraminicacid; PS, polysaccharide. 112,126,127 128,130 92,131-136137 138 139 140,141 142 The ability of a microorganismto produce surface lectins signifies the role of lectinsin the ecology of microbes, especially for biologicalrecognition of host cells [4,17,18,22]. One can also seefrom Table 2 that bacterial lectins are capable ofrecognizing not only several types of monoanddisaccharides (specificities of lectins for simple sugars)or oligosaccharides (fine specificities of lectins), butalso macromolecular glycoconjugates and polysaccharides.From the example of monosaccharides coupled with suitablecarriers it can be observed that the lectin-inhibitingeffectiveness of the neoglycoconjugates shows 10 3 or 106 -fold increase when compared with free carbohydrates[185,186]. The effectiveness of such conjugates dependsnot only on the monosaccharide type, but also on itsdensity and distribution over the carrier surface. Lectinsof Actinomyces are capable of recognizing the samestructures of disaccharide fragments within differenttargets, such as glycolipids and polysaccharides [166-168].Pertussis toxin and elder lectins react with suchglycoproteins as fibrinogen, transferrin, and phosvitin,containing Neu-5-Ac-a2,6-residues of sialic acid (but notNeu-5-Ac-a2,3-residues). Fetuin, containing equal numbersof both types of these sialic acid residues, exhibits ahigher affinity for the toxin than for the elder lectins.Human fibrinogen was characterized by maximal affil).ityfor the toxin. Fibrinogen glycoprotein contains fouridentical biantennary asparagine-bound glycans, withterminal sialic acid residues. More weakly reacting withthe toxin were human transferrin and chicken phosvitin,which possess two biantennary or one triantennarysialylated glycans, respectively. In its ability to reactwith sialoglycoproteins, pertussis toxin could bedistinguished from a sialospecific plant lectin from wheat

embryos [53,54]. Bacteriallectins also frequently show ahigh selectivity for glycolipids (globosides andgangliosides). With these receptors, lectins ofmicroorganisms are often distinguishable from those ofplant or animal origin [10,17]. Whereas cholera toxin hasa maximal affinity for monosialoganglioside GM~> thetetanus, botulinum, and gas gangrene toxins show highaffinities for gangliosides containing three to foursialic acid residues. Here, the increased selectivity ofthe microbial lectin for glycolipids may be enhancedbecause of variations in the structure of the lipidcomponent. Thermolabile enterotoxins of various serotypesof Escherichia coli differ in their levels of affinity fora wide range of gangliosides [92]. Oral streptococcallectins [12] may be considered an example of thehigh-level selectivity of microbial lectins towardpolysaccharides. Of a set of glucans, with varyingcontents of a-1,6; a-1,4; a-3; and a-1,2linkages, thelectin reacted only with those glucans containing morethan 800Jo of the a-1,6 linkages; the maximal affinity forlectins was a glucan containing

Microbial Lectins 309

950Jo of a-1,6 linkages. Only linear glucans of theforegoing type with a

relative molecular mass (M,) of over 5 x 10 5 kDa wereable to induce rapid

aggregation of the streptococci. From Table 2, one can seethat a single

microbial lectin can show not only selective reaction witha definite type

target (glycoprotein, glycolipid, polysaccharide) but canalso recognize gly

coconjugates of various other types. Cholera toxin, forinstance, may ex

hibit a high specificity toward some glycolipids andpolysaccharides [166

168]. The ability of a microbial lectin to reactselectively with glycoconju

gates is determined by the domain and epitope structuresof the lectin

molecule. Epitope organization of the cholera toxincarbohydrate-binding

subunit [187], and the size and structure ofcarbohydrate-binding fragments

or subunits of diphtheria, pertussis, and other microbialprotein toxins

also vary. Filamentous Bordetella hemagglutinin containsbinding sites for

heparin or glycoconjugates containing terminal residues ofgalactose that

are not adjacent to each other [59]. Interaction ofClostridium toxin A with

antibodies does not interfere with hemagglutinatingactivity of this toxin

lectin, which is characterized by a h,igh selectivitytoward complex glycans

[68,69]. Therefore, variations in selectivity ofmicrobiallectins toward gly

coconjugates may occur because of the existence of avariety of sites with

different specificities for glycoconjugates and because ofstructural changes

within these sites. Lectins from actinomycetes,streptomycetes, and Sclero

tium rolfsii interact differently with variousgram-positive and gram-nega

tive bacteria [166,182,183].

IV. PROTOZOAL LECTINS

Currently, protozoallectins have been studied in limiteddetail relative to

interactions with other microorganisms. Lectins fromprotozoa are also

characterized by the aforementioned general regularities:production of a

system of lectins with different carbohydratespecificities by a microorgan

ism of one species or strain and the ability of the samelectin to interact

with various types of carbohydrate-containing targets(Table 3). In addition

to the glycoproteins and neoglycoproteins listed in Table 3that interact

with protozoallectins, there are reports on the interactionbetween Giardia

Iamblia lectin and Salmonella lipopolysaccharides [143].

V. YEAST LECTINS

As with the protozoal lectins, little information isavailable concerning

carbohydrate complexes with yeast lectins [154,155]. Themost thorough

study has been given to the preparations of lectins fromthe pathogenic yeast species Candida a/bicans, the causeof candidiasis in humans, as well as lectins of yeastswidely used in the food industry, produced by strains ofSaccharomyces cerevisiae (Table 4). It is worth mentioningthat the spectrum of biological activities of yeastlectins also includes killer activity toward other yeastspecies. Thus, extracellular lectin from Pichia anomalashows anticandidal activity owing to selective interactionwith {:l-1,6-glucan of C. albicans [160]. Anothersignificant process of biological recognition betweenyeast gametes of different sexes is controlled byparticipation of yeast a-mannan-sensitive lectins [162].Table 3 Interaction of Lectins of Protozoan Origin withGlycoconjugates and PolysaccharidesCarbohydrate-containing Lectin sources targets Ref.Entamoeba histolytica lectin Asialoorosomucoid; Gal,GalNac144 containing polymers of CHO hamster cell lineSurface adhesin N-, 0-glycan of cell surface of CHO145-147 hamster cell line, mucin of rat intestineGiardia Iamblia Portland 1 strain surfaceGlucose/mannose-containing poly148,149 lectin (57-78kDa)

mers of intestinal epidermal cells of mammalsTagerin/lectin activated Mannose-6-phosphate-containingby trypsin (28-30kDa) materials of rabbit erythrocytesLeishmania braziliensis (NR) GlcNAc-BSA,N-acetylglucosamine 150 surface lectin glucose, mannose,galactosecontaining materials of macrophage J774G8 L.donovani transporter of Glucose-containing ligands 91glucose Paramecium tetraurelia lectins Gal-BSA, Man-BSA,dextran-BSA 151 of apex of trichocysts Plasmodiumfalciparum Glycophorins, GlcNAc-Sepharose 152 (140,70,35kDa) Shizont soluble antigen Sialoreceptors of humanerythro153 (175 kDa) of camp strain cytes Pneumocystiscarinii adhesins (j-Gal-BSA, Man-BSA a-Fuc-BSA 110 oramino sugar-BSA

Microbial Lectins 311

Table 4 Interaction of Lectins of Yeast Origin withGlycoconjugates and Polysac

charides Carbohydrate-containing

Lectin sources targets Ref.

Candida albicans extracellular L-Fucose-containingsubstances of 156

lectin of GDH 2346 strain buccal and vaginal epithelialcells of mammals Extracellular lectin ofN-Acetylglucosamine-containing rna157 GDH 2023 strain C.terials of epithelial cells of mamalbicans and Cryptomals.Gal-131, 4-Glc-!31 containing coccus neojormansglycolipids adhesins

K/uyveromyces bulgaricus ex13-GlcNAc,aGal-containingmaterials 158,159

tracellular lectins of sheep and rabbit erythrocytes,yeasts

Pichia anomala extracellular 131 ,6-glucan-containingsubstances of 160

anti candidai toxin cell wall of Candida albicans

Saccharomyces cerevisiae exGalactoseand lactose-sensitivepoly161

tracellular hemagglutinin of mers of animal erythrocytes

CD115 strain Sex agglutinin (22 kDa) Mannose-containingpolymers of 162 yeasts

VI. FUNGAL LECTINS

Lectins of fungi have been less well investigated thanthose of higher plants

[10]. Many of the fungal lectins studied belong to thegroup of chitin

binding proteins [163,164]. Data on the interaction offungallectins with

other carbohydrate-containing targets are presented inTable 5. Many fun

gallectins are characterized by a high affinity forsialomucins of the bovine

submaxillary gland [164,169,170,178], whereas lectins fromAthelia ro/jsii

and Rhizoctonia so/ani mycelia show a high affinity forsialomucins of the

pig intestinal mucous membrane [172]. Some fungallectinsalso interact

well with human erythrocyte glycoproteins, bovine fetuin[164,172], and

glycosaminoglycans of the chondroitin sulfate type [170].Of considerable interest are data on specific interactionof fungal lee

tins with polysaccharides. Lectins from Rhizoctonia so/anireact well with

galactose and N-acetylgalactosamine-containingpolysaccharides of bacte

rial and plant origin [166,181]. Lectins from Phanerochaetechrysosporium

and the bacterium Streptomyces murinus [179, 183] showaffinity for {3Table 5 Interactions of Lectins of FungalOrigin with Glycoconjugates and Polysaccharides ~ ;jLectin sources Chrysosporium keratinophilum (Frey)conidial hemagglutinin Arthrobotrys ellipsospore

extracellular hemagglutinin Arthrobotrys oligosporasurface adhesins Athelia roljsii micellar hemagglutinin(17 kDa) Beauveria bassiana micellar hemagglutininsCephalosporium acremonium extracellularagalactosidase/hemagglutinin Clitocybe geotropa, Laccariaemethystina, and Photo/iota squarrosa lectinsConidiobolus obsurus lectins/adhesins of spore surfaceGanoderma lucidum micellar hemagglutinin (17 kDa)Epidermophyton floccosum, Microsporum canis, M. cookei,and M. fulvum extracellular hemagglutinins Neurosporasitophila extracellular hemagglutinin (22 kDa)Phanerochaete chrysosporium extracellular cellobioseoxidase with domain for sorption on glucan (flavin domainof enzyme) Pythium aphanidermatum lectin Rhizoctoniacrocorum micellar hemagglutinin (11 kDa) R. so/animicellar hemagglutinin (13 kDa) Sclerotium roljsiiextracellular lectins (55+ 60 kDa) Carbohydrate-containingtargets N-Acetylneuraminic acid and Ca 2 + -sensitivesubstances of rabbit erythrocytes, mucin of submandibularbovine gland Mucin of submandibular bovine gland andchondroitin sulfate containing substances of chickenerythrocytes 2-Deoxy-n-glucose substances of cell surfaceof Trichostrongylus colubriformis Mucin of pig intestine;asialofetuin-sensitive substances of trypsin-treatederythrocytes of pigs Monosaccharide-insensitivesubstances of animal erythrocytes Branched {31,4-glucanpotato starch L-Fucose-containing substances of spores,flagella, sporangia and rhizoids of fungal strains fromintestine of sheep Glc-BSA, GlcNAc-BSA Sheep erythrocytesN-Acetylneuraminic acid-containing substances of rabbiterythrocytes; mucin of bovine submandibular gland; GP ofhuman erythrocytes Mu~ of bovine submandibular gland; GPof human erythrocytes {31 ,4-Glucan (cellulose)Fucose-containing substances of Lepidum sativum Mucin ofpig stomach; fetuin-sensitive substances of trypsintreatedrabbit erythrocytes Gum arabic;N-acetylgalactosamine-sensitive substances of rabbiterythrocytes Surface polymers of E. coli Ref. 169 170171 172 173 174 175 176 177 178 163 178 171,179172 181 182

1,4-glucans or ~-1,3(6)-glucans. The lectin ofCephalosporium acremo

nium binds specifically with a-1 ,4-glucan [174]. Amongfungallectins, simi

lar to some lectins from bacteria (see Table 2) are someenzymes, such

as cellobiose oxidase and a-galactosidase, with aspecificity for glucans

[174,179]. Both of these enzymes of carbohydratemetabolism are charac

terized by the presence of carbohydrate-binding sites inaddition to a cata

lytic center [179]. As can be seen from Table 5, lectinsof fungi may be applied to the

investigation of glycoconjugates, polysaccharides, andreceptors of micro

organisms. With the help of fungal fucose-specificlectins, one can detect

considerable structural subtleties between eight strainsof five other fungal

species belonging to the genera: Neocallimastix,Piromonas, and Sphaero

monas [175].

VII. CONCLUSIONS

The foregoing summary was devoted to interaction of lectinsand aggluti

nins from more than 100 microbial sources, withglycoconjugates, polysac

charides, and cellular receptors. Most microorganisms seemcapable of

synthesizing a group (or a system) of lectins characterizedby various carbo

hydrate specificities, various cellular distributions, anda variety of biologi

cal activities. The set of lectins can vary in mutants andrecombinant micro

organisms, and they also depend on the life cycles ofmicroorganisms. The

ability of a microbial lectin to recognize carbohydrates

generally increases

within the range: free monoand disaccharides (simplecarbohydrates <

isolated oligosaccharides < mono-, di-, andoligosaccharides within glyco

conjugates and polysaccharides). Microbial lectins areable not only to rec

ognize various types of natural carbohydrate-containingtargets (glycopro

teins, gangliosides, globosides, lipopolysaccharides,glycosaminoglycans,

and polysaccharides), but they can also recognizedifferences in carbohy

drate-containing targets within each type. Factors that canpotentially af

fect the affinity of a microbial lectin for complexglycans as constituents of

carbohydrate-containing targets may be the following: (1)types of terminal

and internal carbohydrate residues; (2) presence ofterminal residues of

carbohydrates of the same type in clusters, accessible tolectin (nearest

neighbors within one glycan in a glycoconjugate, such as apolysialoganglio

side or sialylated antenna(e) in polyantennaryasparagine-bound glycans of

glycoconjugates. Shark glycans bound with serine orthreonine in glycopro

teins represent another example; (3) the degree ofpolysaccharide branch

ing. Further refinement of lectin specificity to glycanswithin carbohydrate

containing targets may be residues bearing hydrophobicgroups (methyl,

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13. Sharon N. Biomedical aspects of glycoconjugaterecognition. Biomedical aspects of lectins. Biochem SocTrans 1989; 17:11-12.

14. Weir OM. Carbohydrates as recognition molecules ininfection and immunity. FEMS Microbiol Lett 1989;47:331-340.

15. Old DC. Bacterial cell envelopes in adhesion. In:Hancock I, Poxton I, eds. Bacterial cell surfacetechniques. Chichester: John Wiley & Sons, 1988:227240.

16. Ofek I. Lectinophagocytosis mediated by bacterialsurface lectins. Zentralbl Bakteriol Hyg [A1] 1989;270:449-455.

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181. Vranken AM, Van Damme EJ, Allen AK, Peumans W.Purification and properties of a N-acetylgalactoseaminespecific lectin from the plant pathogenic fungusRizoctonia so/ani. FEBS Lett 1987; 216:67-72.

182. Barak B, Chet I. Lectin of Sclerotium roljsii: itspurification and possible function in fungal-fungalinteraction. J Appl Bacteriol1990; 69:101-112.

183. Suzuki K, Kosai M, Yokomizo K, Uyeda M, Shibata M. SAFa new cell aggregation factor produced by Streptomycesmurinus strain no A-2805. Agr Bioi Chern 1987;51:3017-3025.

184. Jungery M, Pasvol G, Newbold C, Weatherall D. Alectin-like receptor is involved in invasion oferythrocytes by Plasmodium falciparum. Proc Natl Acad SciUSA 1983; 80:1018-1022.

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48. Marcus DM. A review of the immunogenic andimmuno-modulatory properties of glycosphingolipids. MolImmunol1984; 21:1083-1091.

49. Bird GWG. The haemagglutinins of Crotalaria striata.Further evidence of similarity of the A and Bagglutinogens. Vox Sang 1956; 1:167-171.

50. Bird GWG. The red cell. Br Med J 1972; 1:293-297.

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52. Springer GF, Tegtmeyer H, Huprikar SV. Anti-N reagentsin eludidation of the genetical basis of human blood-groupMN specificities. Vox Sang 1972; 22:325-343.

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54. Bird GWG, Wingham J. The action of seed and otherreagents on HEMP AS erythrocytes. ActaHaematol1976;55:174-180.

55. Springer GF. T and Tn, general carcinoma autoantigens.Science 1984; 224: 1198-1206.

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