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Received 6-17-91, 10-9-91; accepted 10-10-91

J. frorozool., 39(3), 1992. pp. 378-385 0 1992 by the Society of Protozoologists

Identification of Acanthamoeba at the Generic and Specific Levels Using the Polymerase Chain Reaction

MICHAEL H. VODKIN,*.**.l DANIEL K. HOWE,** GOVINDA S. VISVESVARA*** and GERALD L. McLAUGHLIN** *Department of Veterinary Pathobio/ogy, College of Veterinary Medicine, University of Illinois, Urbana. Illinois 61801.

**Department of Veterinary Pathobiology, School of Veterinary Medicine, Purdue University, West Lafayetre, Indiana 47907 and

***Division of Parasitic Diseases, Center for Infectious Diseases, Centers for Disease Control, Atlanta. Georgia 30333

ABSTRACT. We have adapted the polymerase chain reaction to identify strains of Acantharnoeba. Using computer-assisted analysis, primers were designed from an anonymous repetitive sequence and from published sequences of 18s and 5s ribosomal RNA genes of A. castellanii. Amplification of a short ribosomal DNA target (272 base pairs) at restrictive annealing conditions (>50" C) resulted in a single band that was unique for the genus and distinguished Acanthamoeba from Naegleria. This assay functioned with fresh and formalin-fixed cells as starting material. Amplification of longer targets (4OC-700 base pairs) at less restrictive annealing conditions (147' C) led to more than one band. This multiple banding pattern could reproducibly classify Acanthamoeba at the strain level and was, in certain cases, diagnostic for known pathogenic strains. However, these assays need to be further refined to make them relevant for clinical purposes. Key words. Amoeba, Naegleria. human pathogen, repetitive DNA, ribosomal DNA.

HE genus Acanthamoeba consists of a number of species, T some of which are represented by multiple strains or in- dependent isolates. Several species have been implicated as hu- man pathogens. Current techniques for identifying and spe- ciating Acantharnoeba are time-consuming and laborious.

' To whom correspondence should be addressed. Department of Vet- erinary Pathobiology, School of Veterinary Medicine, Purdue Univer- sity, West Lafayette, Indiana 47907.

Pathogenicity is inferred only if the isolate comes from a symp- tomatic human or if infections can be produced in an animal model system. Interest in certain pathogenic free-living amoe- bae has recently increased because they have been determined t o be threats t o human health [ 181. T h e genus Acanthamoeba is one of several genera of free-living, bacteriophagic amoebae that are facultative parasites of humans. Various Acanthamoeba species, frequently A. castellanii, A. culbertsoni, and A. polypha- ga. can cause keratitis (generally associated with contact lens

VODKIN ET AL.-PCR IDENTIFICATION OF ACANTHAMOEBA 379

Table 1. Acantharnoeba-specific amplimers.

Template Sequence Identifies

Repetitive Strain APOl .for 1 G AATTCCGCTCGGAAGCC APO 1 .rev 440 AACGCTGGTCTGACGCC

18s rDNA Genus ACARNA.for 1383 TCCCCTAGCAGCTTGTG ACARNAxev 1655 GTTAAGGTCTCGTTCGTTA

18s rDNA Pathogen ACARNA.for 1345 CGCGAGGGCGGTTTA ACARNAxev 1830 GCTGGCTAGGCGCGCAG

18s-5.88 ITS Species ACAlKfor 2209 CGGGCTTGTG AGGTCTC ACA58.rev 92 GATGATTCACTGATCCCTG

use), ulcerations of the skin, and granulomatous amoebic en- cephalitis when the central nervous system is invaded [20]. Sur- veillance of environmental samples, diagnosis of human infec- tion, and improved prophylaxis and treatment are priorities for dealing with these organisms. However, surveillance and di- agnosis are difficult because classic morphologic taxonomic cri- teria are generally unable to distinguish pathogenic from con- specific, nonpathogenic strains. Serologic and physiologic typing of strains from human clinical samples is also a problem.

Some successes in strain typing, however, have been reported with restriction fragment length polymorphism (RFLP) analysis of mitochondria1 DNA isolated directly from organelle prepa- rations [4] or purified as circular DNA [2, 251. Alternatively, this analysis can be performed directly from total DNA with the obtained patterns representing one or more classes of re- petitive DNA [3]. Because of the relatively low complexity of protozoan DNA, moderately repeated sequences are visible on a stained gel. These RFLP analyses have been used to type strains and species in both Acanthamoeba and Naegleria [6, 9, 11, 141.

The polymerase chain reaction (PCR) has recently been mod- ified to type organisms at the specific level. Some approaches have required preexisting sequence data [ 191, while others have shown that even arbitrary primers can differentiate a group of organisms by generating banding patterns similar to those in RFLP analysis [ 13,22-241. We noticed independently that these PCR approaches can be adapted to Acanthamoeba, and the results were presented in brief form elsewhere (Vodkin, M. H., Huizinga, H., Visvesvara, G. S. & McLaughlin, G. L. 1990. Typing of pathogenic Naegleria and Acanthamoeba using the polymerase chain reaction. 39th Annual Meeting of the Amer- ican Society of Tropical Medicine and Hygiene. Abstr.). Using amplimers from two classes of repetitive DNA, characteristic band(s) are reproducibly detected for each species of Acanth- amoeba. For one locus with bands of the same mobility, re- striction digestion of the amplified products is able to resolve strain differences at the primary sequence level. Other investi- gators [8] have noted that the ribosomal DNA (rDNA) of Acanthamoeba contains genus-specific insertions. In the current study, judicious selection of primers and amplification condi- tions have allowed amoebae of the family Acanthamoebidae to be distinguished at several taxonomic levels. Additionally, one primer pair appears to distinguish pathogenic from nonpatho- genic Acanthamoeba. In conjunction with a previously pub- lished sequence, Acanthamoeba can be distinguished from Nae- gleria. Our approach, the analysis of multiple loci using PCR, takes advantage of the sensitivity and specificity of PCR to

generate data which may be useful for clinical, ecological, or taxonomic studies of Acanthamoeba.

MATERIALS AND METHODS All Acanthamoeba isolates were obtained from the reference

collection of GSV. They included the known pathogens iden- tified as A. culbertsoni Lilly A- 1 strain; A. lugdunensis L3a and SH 565 strains; A. polyphaga (CDC:0187: 1); and Acanthamoeba spp. CDC:0786: 1, E, and Mc strains, the last four of which were primary isolates. They also included the following environmen- tal isolates, several of which have been determined to be non- pathogenic: A. castellanii ATCC 3001 1 and Neff strains; A. co- mandoni; A. mauritaniensis SH 197, SH 522, and SH 1652 strains; A. rhysodes HR-clone 2 and R4c strains; and Acan- thamoeba sp. A5 strain. Acanthamoeba were grown, and total DNA was extracted from the harvested organisms as previously described [ 141. DNA from A. polyphaga was digested with Eco RI and cloned into lambda ZAP I1 (Stratagene, San Diego, CA). DNA was radiolabeled with dATP3* with a random primer kit (5 Prime + 3 Prime, Inc., Boulder, CO). Radiolabeled genomic DNA from A. polyphaga was used to screen a homologous li- brary for clones derived from repetitive DNA [15]. Plaque- purified virus was rescued as a plasmid construct (pBluescript I1 KS) per the manufacturer's instructions. Purified DNA was sequenced by the dideoxy chain termination method [ 171. One primer pair (Table l), which theoretically had T, of 54"-58" C (calculated by T, = 2' C [A + TI + 4" C [G + C]), was designed from the obtained sequence, and it was searched against the GenBank and EMBL nucleotide databases for homology using the FASTA program of the GCG package [7]. Primers for spe- cific amplification of rDNA sequences of Acanthamoeba were selected from the published sequences of A. castellanii [8, 121 and also in accordance with the above T, criterion. The 18s rDNA primers were similarly chosen on the basis of non-ho- mology to other eukaryotic sequences according to multiple sequence alignment. The 5.8s rDNA reverse primer was chosen from a region conserved among several other protozoan and fungal species in order to maximize the probability that it would hybridize to all species of Acanthamoeba DNA.

In order to test the ability to detect amoebae in a background of tissue, whole brain or eye from euthanized suckling pigs was fixed and stored in 10% buffered formalin for one month. In vitro cultured amoebae were harvested, fixed, and stored in parallel in 10% buffered formalin. Brain or eye tissue was re- moved from formalin and homogenized in a modified PCR buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCI, 0.1 mg/ml pro- teinase K, 0.1% Triton X-100) in a 1: 100 w/v ratio. After for-

380 J. PROTOZOOL., VOL. 39, NO. 3, MAY-JUNE 1992

CGACCGCTCGCCGAGTAC ---------+-------- 498

Fig. 1. Sequence of APO I . The two primer locations are in lower case. The longest open deduced reading frame is indicated with the single letter amino acid code.

malin-fixed amoebae were pelleted by centrifugation, the for- malin was decanted, and the cells were resuspended in modified PCR buffer. Defined numbers of amoebae were added to tissue samples, and the mixtures were incubated overnight at 37" C in order to digest proteins that may have been cross-linked to DNA. Samples were then boiled for 10 rnin with intermittent vortexing and stored at - 20" C until used.

Polymerase chain reaction was performed using GeneAmp reagents from Perkin Elmer Cetus (San Diego, CA) in 50-p1 reactions containing 1.25 U Taq polymerase, ca. 0.1-1 .O ng total genomic DNA or 75 formalin-fixed amoebae with 50 pg of fixed host tissue, and 0.5 to 1.0 p M primer concentrations. Ampli- fications were performed for 30-35 cycles of 1-1.5 rnin at 94" C, 1-1.5 rnin at 46-50" C, and 1-1.5 min at 72" C, with a final elongation step of 5 min at 72" C in either a Coy (Ann Arbor, MI) Model 50 Tempcycler or in a MJR (Watertown, MA) PTC- 100 Thermocycler. Polymerase chain reaction products were electrophoresed in 1.5%-2.0% agarose gels (IBI, Hartford, CT) in Tris/Borate/EDTA buffer (typically 10-1 5 plllane). The gels were stained for 30 rnin in a 0.2 pg/ml solution of ethidium bromide, destained in deionized H,O, and visualized with short- wave ultraviolet light. For subsequent restriction analysis, spe- cific products were purified with Geneclean I1 (Bio 10 1, La Jolla, CA) either directly from the reaction mixture or after excision from an agarose gel.

RESULTS Primers for repetitive DNA. Two classes of repetitive DNA

have been described to date in Acanthamoeba: the 42 kilobase (kb) circular mitochondria1 genome [2] and the 12 kb rDNA transcription unit [5]. A number of plaques was screened from a library of A. polyphaga by hybridizing with total homologous DNA at a low Cof value, which screens for repetitive DNA. One of the clones, APO 1. was more extensively studied. On a Southern blot it hybridizes to a 1.6-kb fragment in the Eco RI digest of total DNA (data not shown). In addition, the amoeba DNA hybridizes only to the insert DNA and not to the vector portion of the molecule. About 500 base pairs (bp) were sequenced* with the aid of a primer adjacent to the insertion site (Fig. 1). One of the six potential reading frames codes for a polypeptide of at least 1 18 amino acids. When the deduced amino acid sequence of this frame was searched for similarity against the database, 17.1% identical residues were found along a 105 amino acid length of a DNA polymerase associated with a fungal mito- chondrion [lo].

* The nucleotide sequence data reported in this paper will appear in the EMBL, GenBank, and DDBJ Nucleotide Sequence Databases under the accession number M65 180.

VODKIN ET AL.-PCR IDENTIFICATION OF ACANTHAMOEBA 38 1

Fig. 2. Amplification of DNA from various Acanthamoeba spp. with APOl primers. Total DNA from various species was separately am- plified for 33 cycles with an annealing temperature of 47" C. An aliquot of the reaction was analyzed by gel electrophoresis. Molecular weight standards (lambda/Hind HI), lane I ; amplified DNA from A. polyphagu (lane 2), A . rhysodes strain R4c (lane 3), A. mauritaniensis SH 522 (lane 4), A. mauritaniensis SH 197 (lane 5) , A . mauritaniensis strain 1652 (lane 6), A. lugdunensis strain L3a (lane 7), and A. lugdunensis strain S H 565 (lane 8).

Amplification at 52" C or above was specific; only A . polypha- ga DNA yielded a fragment with the expected size of 440 bp. However, with a less restrictive annealing condition (47" C), multiple discrete bands were apparent for all species except A. polyphaga (Fig. 2) . A reproducible and distinctive banding pat- tern readily distinguished all the strains that were examined. Some specificity was observed in that Naegleria fowleri, N. gru- beri, and swine DNA did not amplify under the same conditions (data not shown). In parallel experiments, Naegleria DNA was amplified with a primer pair from a previously described se- quence as a control [ 161.

Primers for rDNA. Three sets of primer pairs from sequences within nuclear rRNA genes were evaluated (Table 1). Five prim- ers were selected from the published sequence of the 18s rDNA [8 ] , and one primer was selected from the 5.8s rDNA of A. castellanii [ 121. The sensitivity threshhold of the longer 18s rDNA target in purified total DNA from A . rhysodes was first estimated. Figure 3 shows a four-log dilution series of DNA with amplification of rDNA 1345-1830 with and without a background of 1 pg of calf thymus DNA. Standard PCR assays with 0.3 pg or more of amoeba DNA gave an easily detectable band of the predicted size (485 bp), although both primary and secondary band intensities were altered in the presence of com- plex DNA. The latter results as well as comparisons of profiles from boiled whole cells with those from purified DNA (data not shown) suggest that samples to be compared by PCR fin- gerprinting should be processed in parallel.

Fig. 3. Amplification of varying amounts of purified DNA from A. rhysodes Hr-clone 2. Dilutions of the DNA were amplified at a 46" C annealing temperature with primers that encompassed a 485-bp target ( I 345-1 830) of 18s rDNA. Fifteen microliters of a 50 pl reaction mix were analyzed on a 2.0% agarose gel. Lane I , 300 pg of amoeba DNA in the reaction mix; lane 2, 30 pg; lane 3, 3 pg; lane 4, 0.3 pg; lanes 5- 8 repeat lanes 1 4 , respectively, with the addition of 1 p g of calf thymus DNA. Lane 9, 4x 174/Hae 111 marker DNA.

In the case of an 18s rDNA primer pair (Table l), which spans a relatively short fragment, a unique band of 272 bp was generated under all our assay conditions for 10 species of Acanthamoeba tested (data not shown). We tested whether this short target could serve as a robust genus-specific marker. Nae- gleria fowleri was not detected by this primer pair at 50" C. In Fig. 4, lanes 1 and 2, Acanthamoeba DNA give the expected 272-bp band, but Naegleria DNA does not (lane 3). A primer pair, NF9.for 121 and NF9.rev 320, from a previously described N. fowleri mitochondria1 sequence [ 161, was selected. This prim- er pair was predicted to generate a 199-bp fragment from N. fowleri DNA, and this feature was used as a positive control for detecting Naegleria DNA (lane 6). This primer pair (ACAR- NA.for 1383 and ACARNA.rev 1655) was also tested under conditions that mimicked clinical assays. Formalin-fixed Acanthamoeba and host tissue (eye or brain) were mixed, di- luted, and assayed. The predicted band size (272 bp) appeared in only those sample mixtures that contained Acantharnoeba (Fig. 5).

Primers were also designed to amplify a larger portion of the

382 J. PROTOZOOL., VOL. 39, NO. 3, MAY-JUNE 1992

Fig. 4. Differential detection of Acanthamoeba and Naegleria. In lanes 1-3, ACARNA.for 1383 and ACARNAxev 1655 were used. The primers for lanes 4-6 are NF9 121-141 CTGTTTAAGTAATT- TAGTCGG and NF9 320-300 CITCTATTAATGCCCAAAATG, from the mitochondria1 ATPase 6 subunit [16]. Amplification was for 35 cycles with an annealing temperature of 50" C. Lanes 1 and 4, A . cas- tellanii (ATCC 3001 1); lanes 2 and 5, A. polyphaga; lanes 3 and 6, N. fowlen' (8B2); lane 7, bx 174/Hae 111 marker DNA.

18s rDNA (from 1345- 1830; Table 1) of Acanthamoeba. This larger target produced two distinct patterns after amplification (Fig. 6). The five nonpathogenic species of Acanthamoeba tested (lanes 1-4 and 10) had a prominent band (485 bp) with the predicted size as did one of the five isolates (lane 8) that is considered a pathogen. One of the other four pathogens had a minor band of 485 bp (lane 6); the other three had none of that size (lanes 5, 7, and 9); all four pathogens had several other conserved minor bands. The absence of the expected band in the pathogenic species suggests divergence at either one or both primer sites and thus may be a pathogen-specific trait. An in- ternal oligonucleotide within this region hybridized only to the 485 bp fragment of the non-pathogens and not to the secondary bands of the pathogens (data not shown), indicating that these secondary amplification products probably do not originate from 18s rDNA. Mixing DNA that amplified a prominent band with an equal amount that amplified other targets at a reduced in- tensity still allowed the predicted composite pattern (data not

Fig. 5. Amplification of 1383-1655 18s rDNA target from fixed amoebae in the presence or absence of fixed tissue. Whole brain or eye and in vitro cultured Acanthamoeba were fixed separately in 10Yo buf- fered formalin for 4 wk. Tissue was homogenized in a modified PCR buffer. Known numbers of amoebae in PCR buffer were boiled in the presence or absence of the homogenate of fixed brain or eye tissue. Aliquots of the lysate representing 75 amoebae, with or without 50 pg wet weight of tissue, were amplified directly with an annealing temper- ature of 50" C. Lane 1, A. comandoni with eye tissue; lane 2, A . coman- doni with brain tissue; lane 3, A. polyphaga with eye tissue; lane 4, A. polyphaga with brain tissue; lane 5 , A. comandoni; lane 6, A. polyphaga; lane 7, eye tissue; lane 8, no amoebae or tissue. Lane 9, @x 174/Hae 111 marker DNA.

shown), which suggested that bands of low intensity were not suppressed in a background of a high-intensity band.

The last target included 94 bp of 18s rDNA and 92 bp of 5.8s rDNA (based on A . castellanii) and a species-dependent length of the internal transcribed spacer (ITS), a locus that is expected to evolve more rapidly than the actual rDNA. A prom- inent band of about 600 bp that varied within 100 bp was amplified (Fig. 7); a similar band was observed for N. fowleri but not for vertebrate DNA (data not shown). In at least one case (Acunthamoeba sp. Mc strain, lane 7), a doublet was ob- served. To confirm and exploit the sequence vanation of the products, aliquots of the reaction were subjected to endonu- cleases with tetranucleotide recognition sites. In general, the sum of the molecular weights of the digestion products approximates that of the undigested band. These data are consistent with templates of varying size and/or sequence. The digestion with Taq I is presented in Fig. 7 (lanes 9-14). Results are consistent with one conserved Taq I site at the predicted 35 bp from the

VODKIN ET AL-PCR IDENTIFICATION OF ACANTHAMOEBA 383

Fig. 6. Amplification of 18s rDNA 1345-1830 at restrictive con- ditions (50' C annealing temperature). Lane 1, A. custellanii (ATCC 300 1 1); lane 2, A. cornandoni; lane 3 , A. custellunii Neff strain; lane 4, A. rhysodes Hr-clone 2; lane 5, Acunthurnoebu sp. E strain; lane 6, Acuntharnoeba sp. (CDC0786: 1); lane 7, A . polyphaga; lane 8, Acunth- amoeba sp. Mc strain; lane 9, A . culbertsoni Lilly A-1 strain; lane 10, Acunthurnoeba sp. A5 strain; lane 1 1 is q5x 174/Hae I11 marker DNA. (Lanes 5-9 represent confirmed pathogenic isolates.)

3' end of the 18s rDNA, and presence or absence of a second Taq I site which generates two fragments (lanes 10, 11 and 14) that vary slightly in size depending on the length of the uncut amplified product. The 35-bp band cannot be directly observed in this gel system, but its presence is inferred by the increase in mobility of the original amplification product.

DISCUSSION We devised a PCR-based strategy to detect the presence of

the genus Acanthamoeba and to differentiate its members at the specific and subspecific levels, including pathogen vs. nonpatho- gen. Part of the strategy relied on the selection of repetitive

Fig. 7 . Amplification of Acanthurnmba 18s rDNA 2209-5.8s rDNA 92 and restriction analysis of the PCR product. After amplification, one aliquot of the reaction mix was analyzed by gel electrophoresis. Another aliquot was digested with Taq I and analyzed on the same gel. Lane 2, A. custellunii Neff strain; lane 3, A . cornandoni; lane 4, Acunthumoeba sp. (CDC0786:I); lane 5 , Acunthurnoebu sp. E strain; lane 6, Acunth- amoebu sp. A5 strain; lane 7, Acuntharnoebu sp. Mc strain; lanes 9- 14, respectively, represent the Taq 1 digests in the same order as lanes 2-7. Lanes 1 and 8, $JX 174/Hae 111 marker DNA.

DNA targets to enhance sensitivity. This approach builds on published RFLP analyses of repetitive DNA [2, 4, 6, 9, 1 1, 13, 141. The use of PCR with these repetitive loci, however, has at least three potential advantages over those previous studies. The necessity of culturing organisms from biological samples is by- passed; DNA extractions are no longer required; the sensitivity (in this report, one to 10 organisms) is increased.

No confirmed mitochondria1 DNA sequence data are avail- able for the genus. A portion of an anonymous repetitive DNA, cloned from A . polyphaga, was sequenced to generate data for the selection of primers. The cloned fragment comigrates with and hybridizes to a 1.6-kb Eco RI fragment of Acanthamoeba repetitive DNA. Since APOl is not homologous to rDNA, it is possible that it originated from mitochondria1 DNA, the only other repetitive DNA described at this time.

Nuclear 18s rDNA of one species of Acanthamoeba was al- ready sequenced and was larger than that of almost every other eukaryote [8]. Primers were picked in areas that by multiple sequence alignments did not appear to be generally represented in prokaryotes or other eukaryotes. Such primers are expected to be genus specific, and our results were consistent with this expectation.

384 J. PROTOZOOL., VOL. 39, NO. 3, MAY-JUNE 1992

Each of the primer pairs displayed its own specificity and potential utility (Table 1). A short internal 18s rDNA primer pair produced a 272-bp band at both high and low stringency from all Acanthamoeba species tested. This assay worked with fresh amoebae as well as with material fixed and stored in for- malin, as clinical samples sometimes are. Since no amplification occurred with host tissue alone or with N . Jowleri, a known human pathogen, the 18s rDNA 1383-1655 template is a can- didate to detect the presence of Acanthamoeba selectively and sensitively, especially in human tissue. Another amplimer can detect Naegleria sp. in the presence of Acanthamoeba [ 161.

The size ofthe nuclear rDNA ITS target served to differentiate Acanthamoeba, in many cases, at the species level. Subsequent restriction digestion of the target, without gel purification, re- vealed two populations of ITS. In some cases, they coexisted within the same isolate (Fig. 7, lane lo), with different copy numbers or amplification efficiencies. These data can be inter- preted in a number ofways. Although isolates of pathogens were derived from individual humans, a mixed infection is still pos- sible as an explanation. Because no subsequent cloning was undertaken, a mixed population of ITS could have originated from sampling a mixed population of amoebae and then main- taining that mixed population in the laboratory. The isolates have been passaged many times under laboratory conditions, but we consider it unlikely that cross-contamination occurred in the laboratory. The heterogeneity of the ITS could have ex- isted at the time of isolation or could have arisen while in the laboratory. The heterogeneity could have biological significance; some species of Plasmodium have two populations of rRNA which are differentially expressed in warm-blooded and cold- blooded hosts [2 I]. However, the heterogeneity seems confined to the ITS locus, which does not code for functional rRNA. Among bacteria, polymorphisms at the ITS locus have been shown to be useful for species identification [ l ] (Mcbughlin, G. L., Howe, D. K., Smith, A, R., Ludwinski, P., Fox, B. C., Tripathy. D. N., Frasch. C. E.. Wenger, J. D., Carey, R. B., Hassan-King, M. & Vodkin, M. H., unpubl. data).

The longer 18s rDNA target offers the most promise for dis- tinguishing pathogen from nonpathogen. The predicted band from this primer pair was not visible in three of the five tested isolates considered to be pathogenic and was only barely visible in a fourth pathogenic isolate (Fig. 6). These results did not change even when reaction conditions were varied (data not shown). Its virtual absence, in comparison to the intense signal observed for the five non-pathogenic isolates, suggests that ab- sence of this band may serve as a marker for pathogenic Acanth- amoeba. Simultaneous two-tube reactions with two independent amplimers can be performed to detect Acantharnoeba and assess its pathogenicity. To increase the specificity of future assays, the 18s rDNA and amplified secondary fragments from a patho- genic strain should be sequenced, and candidate primers for robust amplification of pathogens should be evaluated.

The repetitive template, APO 1, displayed the greatest taxo- nomic discrimination. At a high stringency (> 50" C), only DNA from the homologous strain (A. polyphaga) was amplified. At reduced stringency (<47" C), each species and even independent isolates of the same species yielded a distinctive multibanded pattern. Similar phenomena were observed when a long rDNA template was amplified under nonstringent conditions. Others have also noted that single, double, or random anonymous primers can generate complex but discrete banding patterns from a given genome [ 19, 22-24]. These banding patterns have been used to monitor genetic linkage to quantitative traits [ 131. Using defined primer pairs at reduced stringency, these banding patterns may also be useful to type protozoan species complexes

that contain pathogenic members. This approach discriminates below the species level and even distinguishes between strains of the same species. However, multiple isolates from the same location had, in one case, indistinguishable patterns (data not shown). In summary, both defined and partly defined primers have yielded potentially useful information with regard to di- agnosis and classification ofAcanthamoeba and other organisms by PCR. Sequence information from the secondary targets may clarify the rules in this new PCR application and enable primers to be chosen rationally to make the technique even more useful for classification and identification of amoebae.

ACKNOWLEDGMENTS This research was supported by a Public Health Service grant

R 0 1 EY08205 to GLM. Computer support was provided to MHV by the Pittsburgh Supercomputer Center grant PSCB DMB890077P. We thank Vicki Gordon and Flo Brandon for assisting on the growth of the Acanthamoeba, Dr. Gail Scherba for critical comments on the manuscript, and Dr. Harry Hui- zinga for supplying Naegleria DNA.

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Received 5-22-91, 8-9-91, 11-27-91; accepted 1-2-92

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Characterization of the Calcium-Binding Contractile Protein Centrin from Tetraselmis striata (Pleurastrophyceae)

DONALD E. COLINC*J and JEFFREY L. SALISBURY**,Z *Center for NeuroSciences, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106 and

**Laboratory for Cell Biology, Department of Biochemistry and Molecular Biology, Mayo Clinic Foundation, Rochester, Minnesota 55905

ABSTRACT. Centrin is a major protein of the contactile striated flagellar roots of the green alga Tetraselmis striata. We present a newly modified procedure for the preparation ofcentrin in sufficient quantity and purity to allow for detailed biochemical characterization. We establish that centrin purified by differential solubility, followed by phenyl-Sepharose and DEAE-Sephacel chromatography is identical with the protein extracted directly from striated flagellar roots with regard to molecular weight, isoelectric point, and calcium- dependent behavior in SDS-PAGE. We also compare the biochemical properties of purified centrin with calmodulin isolated from Tetrasehis and calmodulin isolated from mammalian brain. Centrin can be fully distinguished from either algal or mammalian calmodulin on the basis of molecular weight, isoelectric point, calcium-dependent behavior in SDS-PAGE, proteolytic peptide maps, amino acid composition, ability to activate bovine brain phosphodiesterase, and reactivity with specific antibodies.

Key words. Calmodulin.

ENTRIN is a 20,000 M, acidic phosphoprotein associated C with basal bodies of flagellated green algae [ 15,251. Several lines of evidence suggest that centrin is a novel calcium-binding contractile protein. The protein was first identified as the major protein o f the striated flagellar root (SFR) of Tetraselmis striata [25]. Striated flagellar roots are calcium sensitive contractile organelles [25, 261. Centrin comprises greater than 63% of the protein extracted from isolated SFR by 6 M urea [25]. Prelim- inary in vitro assembly experiments with urea extracts of SFRs have shown the formation of filamentous centrin-base networks following dialysis against low salt buffers, and the conversion of the filaments t o 16-nm clusters with the addition of calcium [25]. Centrin also exhibits calcium-dependent conformational changes which result both in altered mobility in alkaline urea polyacrylamide gel electrophoresis [25] and binding/elution on hydrophobic chromatography media such as phenyl-Sepharose

I Present address: Kresge Hearing Research Institute, Department of Otolaryngology, The University of Michigan, 1301 E. Ann St., Ann Arbor, Michigan 48 109.

To whom correspondence should be addressed. Abbreviations used: SFR, striated flagellar root; SDS, sodium dodecyl

sulfate; Ig, immunoglobin; EGTA, [ethylenebis(oxyethylene- nitrilo)]tetraacetic acid NP-40, nonidet P-40; PAGE, polyacrylamide gel electrophoresis; PVDF membrane, polyvinylidene difluoride trans- fer membrane.

[5,22]. Treatment of living Tetraselmis cells preadapted to low levels of free calcium with a calcium shock; i.e. elevating external calcium levels to 50 m M results in contraction of centnn-based filaments of SFRs: contraction is mediated by bendingand twist- ing of individual filaments [2 I]. These biochemical and struc- tural observations have led to a novel model for the mechanism for SFR contraction which involves supercoiling of centrin- based filaments upon calcium binding [2 1, 251.

Centrin has been detected in a number of other flagellated green algae by immunofluorescence and Western immunoblot assays with antibodies raised against the Tetraselmis protein [ 17, 231. The green alga Chlamydomonas reinhardtii possess several centrin-based cytoskeletal systems within the same cell, where it is localized in three calcium-sensitive contractile fiber systems: descending fibers, the structural homologue of the SFR [23, 24, 311; the distal fiber interconnecting basal bodies [18, 241; and a stellate fiber system o f the basal body-flagellar tran- sition zone [27]. Additionally, centrin is localized in the mitotic spindle of Chlamydomonas cells [24].

A more general interest in centrin arose from the observations that centrin homologues are present in all eukaryotic cells ex- amined, including ciliates and avian, mouse, and human cells [ l , 2, 9, 10, 231 [Salisbury, unpubl. observ.]. In vertebrate cells, centrin has been shown t o localize to centriolar basal feet, peri- centriolar satellites and a fibrous matrix interconnecting satel- lites as well as a t the mitotic spindle poles [ 1, 21.

Fiber systems recognized by anti-centrin antibodies appear