Microbes and Infection 12 (2010) 89e98www.elsevier.com/locate/micinf

Review

Molecular mechanisms of enterotoxigenic Escherichia coli infection

James M. Fleckenstein a,b,c,*, Philip R. Hardwidge d, George P. Munson e, David A. Rasko f,Halvor Sommerfelt g,h, Hans Steinsland g

a Medicine Service Veterans Affairs Medical Center, Memphis, TN 38104, USAb Department of Medicine, University of Tennessee Health Sciences Center, Memphis, TN 38163, USA

c Department of Molecular Sciences, University of Tennessee Health Sciences Center, Memphis, TN 38163, USAd Department of Microbiology, Molecular Genetics & Immunology, University of Kansas Medical Center, Kansas City, KS 66160 USA

e Department of Microbiology and Immunology, Miller School of Medicine, University of Miami, Miami, FL, USAf Institute for Genome Sciences, Department of Microbiology & Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, USA

g Centre for International Health, P.O. Box 7804, University of Bergen, 5020 Bergen, Norwayh Division of Infectious Disease Control, Norwegian Institute of Public Health, P.O. Box 4404 Nyldalen, 0403 Oslo, Norway

Received 24 August 2009; accepted 24 October 2009

Available online 31 October 2009

Abstract

Enterotoxigenic Escherichia coli (ETEC) are a major cause of diarrheal illness in developing countries, and perennially the most commoncause of traveller’s diarrhea. ETEC constitute a diverse pathotype that elaborate heat-labile and/or heat-stable enterotoxins. Recent molecularpathogenesis studies reveal sophisticated pathogenehost interactions that might be exploited in efforts to prevent these important infections.While vaccine development for these important pathogens remains a formidable challenge, extensive efforts that attempt to exploit new genomicand proteomic technology platforms in discovery of novel targets are presently ongoing.Published by Elsevier Masson SAS.

Keywords: Pathogenesis; Enterotoxins; Enterotoxigenic Escherichia coli; Escherichia coli vaccines; Genomics

1. Introduction

Enterotoxigenic Escherichia coli (ETEC) are a diversegroup of pathogens that have in common the ability to colo-nize the small intestine, where they produce and deliverplasmid-encoded heat-labile (LT) and/or heat-stable (ST)enterotoxins. Collectively, these organisms cause hundreds ofmillions of cases of diarrheal disease each year, particularly indeveloping countries. ETEC are responsible for an estimated300,000-500,000 deaths annually in children under the age offive [1]. These organisms are the most frequent cause oftraveller’s diarrhea, and likewise the diarrheal pathogen thatmost commonly afflicts military personnel deployed to

* Corresponding author. 1030 Jefferson Avenue Research (151), Memphis,

TN 38104, USA. Tel.: þ901 523 8990 x6447. fax: þ901 577 7273.

E-mail address: jflecke1@tennessee.edu (J.M. Fleckenstein).

1286-4579/$ - see front matter Published by Elsevier Masson SAS.

doi:10.1016/j.micinf.2009.10.002

endemic areas. In addition, it appears that these organismscontribute substantially to delayed growth and malnutritionaccompanying repeated bouts of infectious diarrhea, andconversely malnourished children appear to be at higher riskof acquiring ETEC infections [2,3].

2. Clinical manifestations of ETEC infection

Enterotoxigenic Escherichia coli infections are classicallyassociated with acute watery diarrhea. Like clinical cholera,these infections can range from mildly symptomatic to severeprofuse cholera-like watery diarrhea [4] leading to rapiddehydration and prostration within a few hours [5]. Indeed,initial isolates of enterotoxin producing E. coli were recoveredfrom cases of apparent cholera where no Vibrio could beisolated [6,7]. In effect, ETEC cannot be distinguished fromcholera on clinical grounds [8]. In addition to diarrhea, othersigns and symptoms including headache, fever, nausea and

90 J.M. Fleckenstein et al. / Microbes and Infection 12 (2010) 89e98

vomiting are often reported, and some patients may haveprolonged diarrheal illness lasting a week or more [9].

3. Molecular mechanisms of virulence

At a minimum, the enterotoxigenic E. coli must be able toproduce, secrete, and effectively deliver LT and/or ST. Giventhe significant phylogenetic diversity observed among ETECstrains, acquisition of genes encoding these toxins may be oneof few essential elements required for development ofa successful pathogenic clone [10]. However, much remains tobe investigated with respect to the overall pathogenesis ofthese strains, and other virulence factors that may be essentialfor colonization and for successful targeting of toxins to hostcells or which may augment this process have not beensufficiently explored.

3.1. Toxins and their secretion systems

3.1.1. Heat-labile toxinHeat-labile toxin (LT), like the closely related cholera

toxin, is a heterohexameric molecule composed of a pen-tameric B subunit, and a single A subunit. The A subunitconsists of two domains linked by a disulfide bridge: A1, theactive toxin molecule, and A2, a helical portion of the mole-cule that anchors the subunit to the B pentamer [11,12](Fig. 1). Binding of the B subunit to GM1 gangliosidescentered in caveolae on the host cell surface [13] triggers

Fig. 1. Structure of the heat-lablie toxin. (Protein Data Bank entry 1LTB)

from [12].

endocytosis of the holotoxin. The enzymatically active A1portion of the A subunit must be translocated across theintracellular membrane to allosterically interact with ADP-ribosylating factors (ARFs) to ADP-ribosylate Gsa, an intra-cellular guanine nucleotide protein [14]. Inhibition of GsaGTPase activity leads to the constitutive activation of adeny-late cyclase. In turn, increased levels of intracellular cAMPactivate the cystic fibrosis transmembrane regulator (CFTR)chloride channel [15] followed by the ultimate secretion ofelectrolytes and water that lead to diarrhea [4].

Both CT and LT are secreted through the outer membraneof their respective pathogens by a two-step process. In the firststep, N-terminal signal peptides of the subunits are cleavedduring sec-dependent [16] transport across the innermembrane to the periplasm where the monomers assembleinto holotoxin [17]. Secretion across the outer membranerelies on a complex type II secretion apparatus known as thegeneral secretion pathway (GSP) [18]. In some strains, addi-tional genes such as leoA [19], a GTP-binding protein [20]encoded on a pathogenicity island in the prototype H10407strain also modulate LT secretion.

The precise process of LT delivery to ganglioside receptorson the surface of intestinal cells is less clear. Earlier investi-gations suggested that optimal delivery of LT occurs onlywhen the bacteria adhere to target epithelial cells [21], and thatanti-LT antibodies which easily bind free toxin are incapableof neutralizing LT delivered by adherent organisms [22].Interestingly, for many years it was thought that ETEC lackedthe ability to secrete LT. However, much of the LT secreted bythese organisms under laboratory growth conditions remainsassociated with outer membrane vesicles which can enter hostcells via lipid raft dependent endocytosis [23]. Studies havealso suggested that LT and its cognate secretion apparatus[24,25] can cluster or polarize to one end of the bacterium,potentially permitting ETEC to deliver their toxin payload ina highly vectored fashion at the host cell surface [24].

In addition to its role in fluid secretion, LT may elicita variety of effects that benefit the organism. LT down-regu-lates innate host responses including defensins [26], andenhances ETEC adherence to epithelial cells [27] and colo-nization of the small intestine [28].

3.1.2. Heat-stable toxinHeat-stable toxins are small cysteine-rich peptides secreted

by ETEC, which bind to the extracellular domain of guanylylcyclase C (GC-C) on the brush border of intestinal epithelium.These interactions activate the intracellular catalytic domainof guanylyl cyclase leading to the intracellular accumulationof cGMP [29,30]. Increases in cGMP in turn activate cGMP-dependent protein kinase II leading to phosphorylation of thecystic fibrosis transmembrane regulator (CFTR) [31] drivingCl- secretion and inhibition of NaCl absorption followed bynet loss of water through osmotic diarrhea.

Several ST peptides have been identified in human ETECstrains. These include the related GC-C-binding STa (STI)peptides ST-Ia (ST-P) and ST-Ib (ST-H), as well as the unre-lated STb (STII) molecules [32], typically associated with

Table 1

Location of genes encoding known and putative virulence proteins in ETEC

strain H10407.

Plasmid-encoded Plasmid size (kb)

CFA/I operon 94.8

EtpBAC two partner secretion system 94.8

EatA autotransporter 94.8

EAST peptide 94.8

ST 1b (ST-H) 94.8

CexE 94.8

ST 1a (ST-P) 66.7

LT 66.7

Chromosomally encoded Location

Tia adhesin/invasin PAIa-selC

leoA GTP binding protein PAI-selC

TibA autotransporter YeeT

TibC (glycosyltranserase) upstream from TibA

a PAI, pathogenicity island.

91J.M. Fleckenstein et al. / Microbes and Infection 12 (2010) 89e98

porcine strains. However, STb binds to different receptors,does not stimulate production of cyclic nucleotides, and hasnot been clearly linked to human disease [33]. STI moleculesshare a core structure of 13 amino acids containing threedisulfide bonds that are required for the biologic effect(Fig. 2). The crystallographic structure of the active ST-P toxindomain [34], predicts the formation of a hexameric ring withresidues N11eA13 forming a putative GC-C binding regionoriented to the outer surface, and it has been postulated thatthis may promote GC-C clustering and activation [35].

Both ST-H and ST-P are plasmid encoded [36] (Table 1),often in transposons [37], and initially synthesized as 72amino acid precursor molecules containing a 19 residue signalpeptide required for entry into the periplasm via Sec. Export ofSTI peptides through the outer membrane requires the trimericTolC protein exporter [38].

Large epidemiological studies that have distinguishedbetween ST-H and ST-P indicate that ETEC producing ST-Hmay be more pathogenic than the ST-P producers [39]. On theother hand, there seems to be little doubt that ST-P-containingETEC are capable of causing disease in humans, because ST-Pstrains have been found to cause multiple food-relatedoutbreaks of diarrhea in Japan [40]. It is not known whetherthe apparent difference in pathogenic potential between ST-H-and ST-P-producing ETEC is a consequence of biologicaldisparity between the two toxins or if they are but markers ofpathogenicity.

3.1.3. EAST1EAST1, the enteroaggregative heat-stable toxin, shares

structural similarity to STI peptides, and also leads toincreases in cGMP [41,42]. The ast gene encoding EAST1 hasbeen identified, often in multiple copies [43], in a variety ofenteric pathogens including ETEC [44]. Studies in ETEC havedemonstrated that this toxin resides on a mobile element andthat when cloned and expressed in a recombinant background,EAST1 is functionally active in assays of enterotoxin activity[42]. While demonstration of EAST1 involvement in ETEC

Fig. 2. Heat-stable peptides. The predicted structure of the active toxin domain

of the ST-P molecule (C5eC17) as determined by Ozaki, et al. Sulfur atoms on

cysteine residues involved in disulfide bond formation are shown in red

(structure was generated from Protein Data Bank Entry 1etn using Protein

Workshop [99]). The peptide sequences of the mature ST-P and ST-H peptides

are shown (residues highly conserved with other GC-C binding molecules

enclosed), and the core toxic domain is underlined.

pathogenesis remains outstanding, the presence of ast genes inmultiple strains may suggest functional redundancy of toxinswith the capacity to provoke elevated levels of cGMP.

4. Adhesins and colonization factors

Colonization of the small intestine is thought to be anessential virulence trait for ETEC. A variety of different struc-tures encoded either on plasmids or the chromosome of ETEChave been identified as putative adhesins or colonization factors.

4.1. Colonization factors (CFs)

A heterogeneous group of proteinacious surface structuresreferred to as colonization factors (CFs) were among the firstvirulence factors identified in ETEC [45] and remain impor-tant targets for vaccine development [46]. At least 25 differentCFs have been described to date and most are plasmid-enco-ded. These structures have been reviewed in some detailelsewhere [47,48].

CFs are antigenically and structurally diverse. Fimbrial,fibrillar and helical structures have been identified, withlengths ranging from 1-to more than 20 mm [49]. CFA/Ifimbriae are among those studied most intensively. Thesefimbriae consist of approximately 1000 copies of the majorfimbrial subunit CfaB, and one (or few) copies of the CfaEadhesin molecule, located at the distal tip. Assembly of theseorganelles requires dedicated periplasmic chaperone (CfaA)and outer membrane usher (CfaC) proteins encoded on thesame operon. The assembled structures (z1 mm long), areproposed to adopt spring-like helices that can uncoil whensubjected to shear stresses in the gut lumen [50,51]. Theprecise receptors for most of the CFs have not been identified,however, many are thought to bind to glycoprotein conjugateson the surface of host cells [52] .

Interestingly, while colonization factors clearly play animportant role in the pathogenesis of human disease, manystrains do not produce a recognizable CF. Moreover, althougha CF-based vaccine did induce protection against more severe

92 J.M. Fleckenstein et al. / Microbes and Infection 12 (2010) 89e98

diarrhea with CF-homologous strains among US travelers toGuatemala [53], the same vaccine failed to induce protectionagainst ETEC diarrhea in Egyptian children [46]. Studies of theprotective effects of CFs following natural ETEC infection havealso yielded divergent results. Studies in Egypt found that inchildren <18 months of age, CFA/I antibodies, (but not anti-bodies against CS3, CS6, or LT) were associated with an inversecorrelation with the risk of diarrhea caused by ETEC expressingthe homologous CF. Likewise, studies in Bangladesh found thatprior exposure to ETEC prevented subsequent diarrhea due toorganisms of the same CF type [2]. Conversely, an extensivecohort study following children from birth until up to two yearsof age found no attributable contribution of CFs to thesubstantial anti-colonizing immunity that these children expe-rienced following ETEC infection [54].

The different findings in these studies likely reflect differ-ences in study methodology. However, they may also beindicative of the complex biology involving multiple molec-ular events that collectively contribute to intestinal coloniza-tion, and subsequent diarrheal disease. Dissection of theseevents and identification of additional adhesins that contributeto colonization, particularly for strains that do not appear tomake one of the known CFs, could yield additional targets forvaccine development.

4.2. Other fimbrial operons

4.2.1. Type I fimbriaeETEC like other E. coli produce common type I fimbriae

[55]. Despite the clear role of these structures in the patho-genesis of uropathogenic E. coli, early studies failed to supporttheir involvement in ETEC virulence [56]. However, subse-quent studies implicating variations of the type I fimbrial tipadhesin, FimH, in tissue tropism [57] raise the possibility thatsome type I fimbriae could be for optimally suited for smallintestinal colonization.

4.2.2. E. coli common pilusOther structures common to many E. coli, including both

commensal and pathogenic isolates, are the E. coli commonpili (ECP) [58]. A potential ECP operon of six genes encodesputative proteins similar to other molecules known to beinvolved in pilus assembly. Recent studies in ETEC demon-strate that approximately 80% of strains carry the ecpA geneencoding the major pilus structural subunit, and that more thanhalf of isolates tested could be shown to produce the EcpAprotein by immunoblotting [59]. While the role of ECP inETEC pathogenesis is not yet established, the high degree ofconservation in this genetically diverse pathotype certainlymake these pili compelling structures for future investigation.

4.3. Non-fimbrial adhesins/invasins of ETEC

4.3.1. Tia and TibA invasinsWhile investigating Salmonella typhi invasion, Elsinghorst

serendipitously discovered that ETEC strain H10407 couldefficiently enter (but not replicate in) cell lines derived from

gastrointestinal epithelium [60]. He then cloned two chro-mosomally encoded toxigenic invasion loci: A (tia) and B(tib). Subsequent investigation revealed that these loci encodedistinct proteins.

Tia, is a 25 kD outer membrane protein [61] encoded ona large pathogenicity island inserted in the selC tRNA gene ofH10407. Tia interacts with host cell surface proteoglycans[62], and by itself is sufficient to promote adherence andepithelial cell invasion when cloned into laboratory strains ofE. coli.

The tib locus encodes TibA an autotransporter (AT) proteinwith homology to several AT adhesins from other mucosalpathogens [63]. TibA is synthesized as a 100 kD precursorprotein, preTibA, that is glycosylated through the action ofTibC [64], a putative glycosyltransferase.

The role played by actual invasion of epithelial cells tomolecular pathogenesis of ETEC remains uncertain. Althoughex vivo studies with human enterocytes from biopsies nicelydemonstrated interaction of ETEC with the brush border [65],similar data from infected patients are lacking. Nevertheless,the ability of Tia and TibA to promote adhesion in vitro andtheir significant homology with known virulence proteins fromother pathogens might suggest that they promote adherence.

4.3.2. EtpAA search for novel secreted proteins led to the identification

of a two-partner secretion (TPS) locus encoded on the largevirulence plasmid of ETEC H10407 [66]. This locus encodesthree proteins: EtpA, a 170 kDa secreted glycoprotein, EtpBa transport pore, and EtpC, a putative glycosyltransferaserequired both for optimal secretion and glycosylation of EtpA.EtpA, like several other members of a growing family of TPSexoproteins, functions as an adhesin. EtpA appears to act ina novel fashion. Recent studies suggest that EtpA functionsa molecular bridge, binding both to host cell receptors and tothe tips of ETEC flagella, where it interacts with highlyconserved regions of flagellin proteins [67]. EtpA, as well asits interactions with flagellin, are required for optimal adhe-sion of H10407 in vitro, and for intestinal colonization ina murine model [68].

Similarity of EtpA to filamentous hemagglutinin, a highmolecular weight TPS adhesin of Bordetella pertussis that isa component of the acellular pertussis vaccine, has stimulatedinvestigation of EtpA as an immunogen. Indeed, vaccinationof mice with either a truncated recombinant 110 kDa EtpAframent [68] or the full length EtpA glycoprotein [69], affordssignificant protection against colonization with ETEC sug-gesting that this molecule could represent a viable target forvaccine development.

5. The importance of flagella to human ETECpathogenesis

E. coli, including ETEC, produce peritrichous flagella eachof which are comprised of an estimated 20,000 individualflagellin molecules. Flagellin molecules stack in a helicalarray in which highly conserved amino and carboxy terminal

93J.M. Fleckenstein et al. / Microbes and Infection 12 (2010) 89e98

regions interact to align inside the flagellar shaft while variableregions orient to the outer surface where they account for ‘‘H’’antigen serotype specificity. Most human ETEC strains aremotile, and the majority of ETEC strains can be serotyped onthe basis of their flagellar H antigens, suggesting that flagellaare likely to be involved in ETEC pathogenesis [70]. However,only recently have these structures been investigated in ETECvirulence. To date, studies have demonstrated that intactflagellar structures are essential for both ETEC adherence andheat-labile toxin delivery in vitro [24], and contribute signifi-cantly to intestinal colonization [67].

Analysis of over 700 ETEC strains identified more than 30different H serotypes, leading to the suggestion that flagellawould not be good vaccine targets unless a conserved epitopecould be identified [70]. The surprising finding that highlyconserved regions of these molecules are exposed at the tips ofsome ETEC flagella [67] and participate in adherence, hasopened the possibility that these domains could be targeted ina vaccine. Recent studies demonstrating cross-protection byimmunization with a recombinant heterologous H antigenappear to support this hypothesis [69].

6. Other potential virulence factors

6.1. EatA

Eat A is an autotransporter encoded on the large virulenceplasmid of ETEC strain H10407 [71]. Genes similar or iden-tical to eatA are present in multiple ETEC strains by DNAhybridization and/or PCR studies, and eatA has also beenfound through genomic sequencing of E24377A [72], and thepCOO virulence plasmid [73]. Interestingly, sequences withEatA homology have also been reported in sequencing ofporcine ETEC plasmids [74]. EatA has been shown to functionas a serine protease, and therefore belongs to a family ofputative virulence proteins known as SPATE proteins (SerineProtease Autotransporters of Enterobacteriaciae). EatA shares

Table 2

Characteristics of ETEC genome sequencing projects.

Strain Serotype Virulence factors Genome

status

Plasmid

descript

E24377A 0139:H28 LT,ST; CS1,CS3 Complete pE2437

pE2437

pE2437

pE2437

pE2437

pE2437

H10407 O78:H11 LT,ST; CFA/I Complete H10407

H10407

H10407

H10407

B7A O148:H28 LT/ST CS6 Draft

(198 contigs)

Yesa

Plasmid only projects

C921b-1 O6:H16 LTþ;STþ; CS1þCS3þ e pCOO

E1392/75 O6:H16 LTþ;STþ; CS1þCS3þ e

a Draft sequences contain contigs that appear to be of plasmid origin, however n

considerable homology with the Shigella flexneriautotransporter, SepA. The precise function that EatA plays inpathogenesis or the actual substrate for its protease activity isnot clear. However initial studies in ileal loops suggested thatEatA could contribute to accelerated virulence of eatAþ strains[71].

7. Genetics of ETEC

7.1. ETEC genomics

With the decreased cost and increased throughput of nextgeneration sequencing technology it has been possible tosequence multiple isolates of any species or pathotype. Thisinflux of genomic data has been applied to E. coli in generaland specifically to the ETEC pathotype on a small scale todate, but considering the importance of this pathogen in globalpublic health we anticipate that tens, if not hundreds ofisolates will be sequenced in the near future. Two genomeshave been completely sequenced and closed including theplasmids (E24377A and H10407) and one other isolate hasbeen sequenced to draft level coverage (B7A) (Table 2).Examination of the genomic content of the ETEC genomeshas revealed that w4% of the E24377A chromosome iscomposed of mobile genetic elements and similar proportionsare observed in the two other genomes [72]. Additionally,when the E24377A and B7A genomes were compared to 15other E. coli genomes, from various pathotypes, on averagethey contained the highest number of unique genes per strain[72]. The combination of a large percentage of mobileelements and unique genes in the ETEC genomes suggests thatthere is a great deal of gene flux in these pathogens.

One very interesting finding to come out of the genomeprojects is the identification of multiple plasmids. Yamamotoand Yokota previously described multiple plasmids in H10407,however there appeared to be variability in the plasmid profilesof isolates from different sources [75]. The sequence data of

ion

Size (kb) GenBank accession Reference

7A_5 5.1 CP000800 (chrom), CP000801 (5),

CP000798 (6), CP000796 (35),

CP000797 (70), CP000799 (74),

CP000795 (79)

[64]

7A_6 6.2

7A_35 35.3

7A_70 70.6

7A_74 74.2

7A_79 79.2

_p52 5.2 http://www.sanger.ac.uk/

Projects/E_coli_H10407/

Not published

_p58 5.8

_p666 66.6

_p948 94.8

AAJT00000000 [64]

94.8 CR942285 [65]

Draft http://www.sanger.ac.uk/Projects/ Not published

o plasmids have been closed from these projects.

94 J.M. Fleckenstein et al. / Microbes and Infection 12 (2010) 89e98

ETEC plasmids suggests that this variability is linked to theidentical mobile elements in the plasmids and chromosomespotentially leading to plasmid instability. Sequencing of thepCOO plasmid by Froehlich et al. revealed that the plasmid isa mosaic cointegrate composed of w24% mobile elementsand two distinct replication origins related to R64 and R100[73]. The mobile elements that litter the pCOO plasmid aswell as the plasmids of E24377A and H10407 are of a fewdefined types including IS1, IS2, IS66, IS629 and IS91. Theincreased proportion of these IS elements in the plasmids andchromosome may allow for integration and resolution of theplasmids in the chromosome at specific points in the ETEClife cycle and contribute to the observed instability.

While currently available genetic information is alreadybeing exploited in ETEC pathogenesis studies, expansion ofthese data to include additional isolates should yield additionaldetails pertinent to understanding the virulence of thesepathogens, and permit detailed exploration of regulatorynetworks. Likewise, given the plasticity of these pathogens,additional genetic information from multiple strains will likelybe crucial to successful identification of conserved vaccinetargets, and should also aid in identification of specific viru-lence factors that may be associated with more aggressiveforms of clinical ETEC infection including severe cholera-likediarrhea that leads to rapid dehydration.

7.2. Genetic/phenotypic diversity and its implicationsfor vaccine development

ETEC represent a phenotypically and genotypically diversegroup of E. coli. This is exemplified by the fact that serotypeanalysis of over 700 ETEC strains isolated from different partsof the world identified 78 different O serogroups and 34different H serogroups in 118 different combinations [70]. Themost commonly identified CF in that study, CFA/I, was rep-resented in 23 different O serogroups.

Results from phylogenetic studies indicate that ETEC haveemerged from the E. coli population on several occasionsthrough independent acquisitions of the enterotoxin genes[10]. Initial chromosomal gene content comparisons of threerecently sequenced ETEC strains stemming from threedifferent ETEC lineages did not result in the identification ofany ETEC-specific genes [10], potentially indicating thatrelatively few chromosomal genes are needed to establish anETEC lineage. However, further studies are clearly needed tomore adequately identify genes that may be shared betweenETEC isolates.

Although strains stemming from different ETEC lineagesmay appear at present to have few obvious antigens incommon with each other except for the toxins and CFs, thereare indications that as yet undefined plasmid or chromoso-mally-encoded antigens may play an important role in thedevelopment of natural immunity against ETEC. In a longitu-dinal epidemiological study of 200 young children in WestAfrica, natural ETEC infections resulted in a 47% protectionagainst new infections with ETEC that carried the samecombination of toxins and CFs, but this protection did not

seem to be attributed to the toxins or the CFs [54]. Strains thathave the same toxin and CF combination often stem from thesame ETEC lineage [76e78], potentially indicating that otherplasmid or chromosomally-encoded antigens may provideprotection against other ETEC that belong to the same lineage.There is currently little known about which ETEC lineagesexist in the human population, or about the rate at whichlineages emerge and disappear from the E. coli population. Forlineage-specific vaccines to become a viable option for thevaccine development effort, attention would probably have tobe focused on protecting against strains from the mostpredominant and stable ETEC lineages.

7.3. Transcriptional regulation in ETEC

As with other enteric pathogens ETEC are likely pro-grammed to respond to multiple environmental signals tocoordinate the expression of virulence factors. For instance,expression of enterotoxins and some pilus types is influencedby the availability of certain carbon compounds, such asglucose. LT production is elevated in the presence of glucose[79] an effect known as catabolite activation that requirescAMP receptor protein (CRP). The consensus DNA bindingsite for CRP is a 22 bp spaced inverted repeat and as its nameimplies, DNA binding is cAMP dependent. The LT promoteris repressed by CRP when it occupies a site centered at �31.5,relative to the transcription start site. When bound to this siteCRP occludes RNA polymerase from the promoter and thusrepresses transcription of the genes encoding LT. Repressionof the LT promoter is relieved by glucose which prevents CRPfrom binding DNA by inhibiting the synthesis of cAMP. Incontrast to LT, CRP activates the expression of ST-H [79].

It is also likely that CRP activates the expression ofa variety of pili including CFA/I, because it has been shownthat their expression is inhibited by glucose (cataboliterepression) [80]. These pili are also positively regulated byRns (CfaD) and all Rns-dependent pilin promoters havea binding site immediately upstream of the promoter’s �35hexamer [81]. Most promoters also have one or more addi-tional Rns binding sites further upstream of the promoter-proximal site. It is not yet known if CRP regulates the pilinpromoters directly or indirectly by controlling the expressionof Rns. For 987P pili, which are expressed by some strains ofporcine ETEC, regulation is indirect with CRP activating theexpression of FasH (FapR) which then activates the piluspromoter [82].

Additional factors likely modulate the expression of viru-lence genes in ETEC. Both LT expression and the productionof some pili, including CFA/I [83]are also repressed by H-NS,a nucleoid associated protein of gram-negative bacteria anda global modulator of gene expression [84].

Interestingly, both CRP and H-NS have been shown togovern the production of flagella in E. coli [85]. Given therequirement of these structures for effective bacterial adher-ence and intestinal colonization [67] as well as toxin delivery[24], it is likely that their expression is coordinated withvirulence factor production in ETEC. This view appears to be

95J.M. Fleckenstein et al. / Microbes and Infection 12 (2010) 89e98

supported by the recent identification of a CRP binding siteupstream of the etpBAC promoter by DNase I footprinting(Fleckenstein and Munson, unpublished).

Searches for additional genes that might be part of the sametranscriptional network can also help to identify novel viru-lence factors. Recently, in silico scanning of sequenced ETECgenomes for putative Rns binding sites was employed toidentify another potential secreted virulence protein of ETEC,CexE [81], encoded on the large 94.8 kB virulence plasmid ofH10407 along with genes for EtpA, EatA, and CFA/I. Inter-estingly, an insertion in cexE was also identified using TnphoAmutagenesis to screen for secreted proteins (Fleckenstein,unpublished), further suggesting that cexE does encode a novelsecreted protein. Rns (CfaD) activates the expression of cexE.Similar to what has been shown for the CS1 pilin promoter,activation of the cexE promoter requires a Rns binding siteimmediately upstream of the promoter’s -35 hexamer. Occu-pancy of an additional, promoter-distal binding site is requiredfor full activation of the cexE promoter. The cexE geneencodes a 12.6 kDa protein whose first 19 amino acidsconstitute a signal peptide that is cleaved during sec-depen-dent transport across the inner membrane. The function ofCexE has yet to be elucidated. However, the location of cexEon a virulence plasmid where its expression is regulated byRns, known to regulate the expression of other virulencefactors, strongly suggests that this gene encodes a novelsecreted virulence protein.

8. Comparisons to porcine ETEC

Enterotoxigenic E. coli are a significant cause of post-weaning diarrhea (PWD) in pigs [86], and similar to humanstrains, ETEC that cause PWD express heat-labile (LT) and/orheat-stable enterotoxins (STb). These organisms also parallelhuman strains in that most express fimbrial adhesins respon-sible for colonization of the small intestine [87]. Despite theseoverall similarities, ETEC in general exhibit considerable hostspecificity, likely due to distinct adhesin/host receptor rela-tionships. Therefore to date, attempts to cause disease withhuman strains in porcine infection models have met withlimited success.

Still, porcine ETEC can provide important clues relevant tothe understanding and prevention of human infections. Indeed,many of the seminal discoveries for ETEC in general weremade in investigation of porcine infections, and ETEC werefirst established as a cause of disease in pigs [86,88], prior toidentification of toxin-producing E. coli as a cause of humandiarrheal disease [89]. Porcine ETEC strains were first used toshow that strains possessing heat-labile toxin activity (lateridentified as LT) were responsible for causing diarrhea invarious animals, and that fimbrial structures (pili) wereinvolved in colonization of the small intestine [87]. Likewise,the present effort toward fimbriae-based vaccines for humanETEC stems in part from early demonstrations that vaccina-tion pregnant swine with fimbriae prevented infections innewborn pigs [90].

A number of important general insights about bacterialvirulence and enterotoxin structure and function have beengleaned from previous and ongoing studies of porcine ETECincluding a determination of the LT subunit composition [91]and its binding specificity for GM1 [92]. Of recent interest hasbeen the concept that bacterial pathogens disseminate viru-lence factors including LT in vesicles released from the outermembrane [23], a process described some time ago usingporcine [93] ETEC isolates.

Recent studies using porcine intestinal epithelial cellcultures [94], have further suggested that the pathogenesis ofETEC in general may be more complex than previouslyappreciated. Interestingly, in this system, LT appears toprecondition the host intestinal epithelium for ETEC adher-ence, in a process dependent upon the ADP-ribosylationactivity of this toxin [27]. Conversely, at least some ETEChave the capacity to induce apoptosis through a processindependent of LT expression [95], and intriguingly thesechanges also appeared to promote bacterial adherence. Whilethe full complement of changes to the host in response tointeractions with ETEC remain to be elucidated, the inter-play of ETEC induced apoptosis of intestinal cells andenhanced adherence is intriguing. These findings shouldstimulate additional clinical investigation particularly inlight of the interplay between ETEC infections and malnu-trition [3].

9. Host factors influencing ETEC infection

Certainly, many parameters including expression ofspecific virulence factors by the bacteria, both innate andacquired host defenses, and the genetic background of thehost influence the clinical presentation of ETEC infectionfollowing ingestion of a sufficient inoculum. However, onlyrecently have studies begun to examine host factors associ-ated with either the acquisition of ETEC infections ordifferent clinical presentations that range from asymptomaticcolonization to severe, life-threatenting diarrhea. As previousstudies showed that individuals with type O blood were atenhanced risk for severe Vibrio cholerae infection, severalinvestigations have now focused on the relationship of ABOblood group determinants to ETEC infections. In contrast toV. cholerae infections, data from Bangladeshi childrendemonstrated no association with blood group O and diar-rhea [96]. However, a subsequent study conducted in a birthcohort of over 300 children in Dhaka demonstrated thatblood groups A or AB were associated with a significantlyhigher prevalence of ETEC diarrhea than those with group Oblood [2]. Likewise, children in Bangladesh with Lewisblood group antigen-a positivity (LeaþLeb�) morefrequently had symptomatic ETEC infections [97]. Presum-ably glycoconjugates on the surface of red blood cells mayalso be expressed on intestinal epithelia where they act asreceptors from one or more bacterial adhesins, and indeedCfaB, the major subunit of Cfa/I fimbriae binds to glyco-sphingolipids including Lea [52]. In addition to the associ-ations with blood group antigens, studies of traveler’s

96 J.M. Fleckenstein et al. / Microbes and Infection 12 (2010) 89e98

diarrhea have also identified a synonymous single nucleotidepolymorphism in the human lactoferrin gene that was asso-ciated with an increased risk of diarrheal illness [98].

It is likely that over time a number of polymorphisms in thehuman genome will be associated with variant clinical formsof ETEC infection, and likewise that specific virulence factorswill be associated with more severe forms of disease. Under-standing these associations could theoretically facilitatedevelopment of vaccines designed specifically to prevent life-threatening diarrheal illness.

10. Future directions

Recent ETEC pathogenesis studies have revealed anincreasing degree of complexity for pathogens that were oncebelieved to simply release their toxin payloads by lysis in thesmall intestine. Exploitation of newly discovered virulencefactors, together with expanded information on ETECgenomes, and anxiously anticipated antigen discovery toolsincluding pan-genome DNA and protein microarrays currentlyin development offer unprecedented opportunities to addressthe challenge of developing a broadly protective vaccine forthese important pathogens.

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