Microbial Pathogenesis 1992 ; 12 : 245-254

Mini-review Adherence and colonization mechanisms of enteropathogenic and enterohemorrhagic Escherichia Coli

Vernon L . Tesh and Alison D . O'Brien

Department of Microbiology, Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814, U .S .A .

Introduction

The enteropathogenic and enterohemorrhagic Escherichia coli Enteropathogenic Escherichia co/i (EPEC) are the causative agents of infantile diarrhea, a persistent watery diarrhea which continues to be a major health problem, particularly in underdeveloped countries . Unlike enterotoxigenic E. coli, EPEC strains do not elaborate heat-labile or heat-stable enterotoxins . EPEC can be distinguished from enteroinvasive E. coli based on the inability of EPEC to mediate keratoconjunctivitis in the guinea-pig conjunctival sac (Sereny negative) . Infection with EPEC is associated with characteristic ultrastructural lesions in the intestine . By electron microscopy, the lesions show a dissolution of microvilli with bacteria intimately associated with the surface of the intestinal epithelial cells and are referred to as attaching and effacing (A/E) lesions .' ' 2 Bacterial attachment is accompanied by an unusual morphologic alteration at the epithelial cell surface, such that the adherent bacteria appear to be attached to the cells at raised, cup-!ike projections called pedestals . Pedestal formation appears to involve the disorganization of the cytoskeleton at regions adjacent to the bacteria . Early epidemiological and serological studies which characterized the EPEC have been reviewed elsewhere ."' Infection with enterohemorrhagic E . coli (EHEC) is associated with outbreaks of hemorrhagic colitis and the subsequent development of serious complications, including the hemolytic uremic syndrome and seizures . Hemorrhagic colitis is characterized by bloody, purulent diarrhea . Hemorrhagic colitis occurs most frequently in developed countries and may be associated with the ingestion of contaminated meats or dairy products . EHEC do not produce heat-labile or heat-stable enterotoxins and fail to elicit keratoconjunctivitis in guinea-pigs . Escherichia co/i serotype 0157 : H7 is the most frequent EHEC isolate .' EHEC have been shown to share with EPEC the capacity to cause A/E lesions with pedestal formation and disarray of the epithelial cell cytoskeleton in gnotobiotic piglets,` infant rabbits"' and chickens ." EHEC strains are lysogenized with one or two bacteriophage(s) which encode the structural genes for the Shiga-like toxins ." Shiga-like toxins probably act to exacerbate damage to the colonic epithelium and mesenteric blood vessels, resulting in the bloody, edematous lesions which are pathognomonic for hemorrhagic colitis . 12 0882-4010/92/040245+10 $03 .00/0

(i) 1992 Academic Press Limited



V . L . Tesh and A . D . O'Brien

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Mechanisms of adherence Among the earliest studies to demonstrate the importance of EPEC adherence and colonization in the pathogenesis of infantile diarrhea was the work of Koya and coworkers, who demonstrated that E. coli 0111 caused diarrhea when inoculated intraduodenally in humans, but not when injected into the large intestine . 1314 Given the fact that the small intestine is not normally colonized with large numbers of coliform bacteria, these data suggested that EPEC must express specialized adherence factors to promote adherence to small intestine epithelial cells . Cravioto and co-workers noted that 80% of the EPEC strains they examined adhered to H Ep-2 (human laryngeal epidermoid carcinoma) cells in vitro in a mannose-resistant manner . 15 Using HEp-2 and HeLa (human cervical adenocarcinoma) cell lines, several investigators demonstrated that EPEC adhered to the cells in two distinct patterns, termed localized and diffuse adherence ." - " Most EPEC strains utilized the localized adherence mechanism, in which the bacteria appeared to bind to a limited number of receptor sites clustered on the target cell surface, producing the so-called microcolony as observed in the light microscope . The diffuse adherence phenotype was characterized by the even distribution of EPEC over the entire surface of infected cells . Both chromosomally encoded 20 and plasmid encoded adherence factors" have been cloned from diffusely adherent EPEC . The importance of diffusely adherent EPEC strains in causing diarrhea remains controversial It was shown that 31 of 32 HEp-2 localized adherent EPEC strains harbored high molecular weight (50-70 MDa) plasmids 25 (Table 1) . Loss of the plasmid resulted in the loss of HEp-2 localized adherence . The transformation or conjugal transfer of the plasmid into an E. co/i K-12 strain conferred upon the non-adherent recipient the capacity to bind to HEp-2 cells . Furthermore, maintenance of the plasmid in an EPEC isolate was essential in mediating efficient adherence in the porcine intestine .2' The capacity of the bacteria to cause diarrhea in volunteers was attenuated in the absence of the plasmid . 26 The plasmid-encoded virulence factor(s) mediating adherence to HEp-2 cells in vitro and to porcine intestinal cells in vivo was termed EPEC adherence factor(s) (EAF) . Using a 1 .0 kb DNA probe derived from the EPEC large virulence plasmid of E. coil 0127 : H6, Nataro et a/ . screened 109 E. coli isolates from Peruvian children and demonstrated that all the EPEC strains which displayed localized Table 1

Localization of EPEC and EHEC virulence determinants

E. coli group EPEC

Plasmid

Chromosome

Factors mediating and/or regulating localized adherence"

Factors mediating diffuse adherence 20

Factors mediating diffuse adherence 21

eaea-

Phage Not known

47

invasion"

Regulation of eae49 Host specificity of lesion formation 51 Regulation of invasion 60 EHEC

Factors mediating and/or regulating adherence

eaea.5n

in vitro" Factors for efficient colonization 31,38 'Associated with actin condensation and A/E lesion formation . References are given as superscripts .

Shiga-like toxins 11



Adhesion and colonization by EPEC and EHEC

247

adherence to HEp-2 cells were EAF+ . Diffusely adhering strains showed no reactivity with the probe . 27 Subsequent studies revealed that factors important in mediating localized or diffuse adherence were encoded on distinct plasmids . 28 The genes gene products on the large plasmids contributing to localized adherence have been characterized . Scotland et al. reported that there were no distinctive fimbriae associated with EPEC, 29 however, by the use of a ruthenium red staining technique, Knutton and

and not EAF co-

workers demonstrated that HEp-2-adherent EAF+ EPEC possessed fimbriae ." This discrepancy may be due to the inducible nature of EPEC adherence factors . VuopioVarkila and Schoolnik demonstrated that the localized adherence phenomenon was induced 30-60 min after transfer of EPEC to HEp-2 monolayers ." Surprisingly, the localized adherence pattern could also be induced in the absence of cells by growing the bacteria in a defined tissue culture medium rather than in a complex enriched medium prior to transfer to cells . Growth in defined medium also resulted in EPEC autoaggregation, supporting the concept that individual EPEC may not attach to epithelial cells ; rather, pre-formed microcolonies may adhere to cells . The selective advantage of such a mechanism of adherence and the environmental signals necessary to induce aggregation in vivo are, at present, unclear . Microcolony formation on HEp2 cells and the autoaggregation phenomenon in defined media by five different EPEC serogroups were shown to be associated with the expression of peculiar cell surface filaments which were 50-500 nm wide, 15-20 hm long and readily interdigitated with one another . 32 These filaments were referred to as bundle-forming pili (BFP) . BFP expression was correlated with the presence of the EAF plasmid ; when two plasmidcured EPEC strains were grown under BFP-inducing conditions, neither the pili nor the localized adherence phenotype were observed . BFP were composed of 19 .5 kDa subunits which shared NH 2 -terminal amino acid sequence homology with the toxinco-regulated pili of Vibrio cholerae . BFP were clearly morphologically dissimilar to the rigid, straight, 7 nm wide fimbriae described by Knutton et al . 30 Although numerous epidemiologic studies have shown that outbreaks of infantile diarrhea are associated with infection with classical EAF' EPEC serotypes, it is becoming increasingly clear that 'non-classical' EPEC strains (i .e . serogroups not normally classified as EPEC, EAF EPEC strains, and strains showing low levels or delayed localized adherence), may be isolated from patients with diarrhea . 33 .34 Carefully controlled epidemiologic studies will be necessary to define the prevalence and severity of infection with these EPEC isolates . Much less is known about EHEC adherence mechanisms . EHEC strains contain large plasmids of approximately 60 MDa which encode structural and/or regulatory genes for fimbriae that are important in mediating attachment to Henle 407 human intestinal epithelial cells, but not to HEp-2 cells or to a panel of human and animal erythrocytes in 3 h adherence assays . 35 Some EHEC strains will, however, adhere to HEp-2 cells with a localized adherence pattern after 6 h of incubation (M . L. McKee, personal communication) . In contrast to the EHEC microcolonies seen on the surface of HEp-2 cells, the EHEC pattern of adherence on Henle 407 cells was less dense, with only 2 to 4 bacteria attached to each cell . Disaggregated fimbriae were characterized by SDS-PAGE and the apparent molecular weight of the integral fimbrial subunit was estimated to be 16 kDa . 35 The presence or absence of the 60 M Da plasmid in E. co/i 0157 : H7 strain 933 did not appear to be important in mediating bacterial adherence in the cecum and colon of gnotobiotic piglets . 36 Wadolkowski et al. utilized a murine model of EHEC infection to study the importance of plasmid-encoded Co litis . 37,38 When streptomycinadherence factors in the development of hemorrhagic treated mice were individually fed either E. co/i 0157 : H7 strain 933 or its plasmid-



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V . L . Tesh and A . D . O'Brien

cured derivative, both strains stably colonized the mice . These data appeared to be in accordance with the earlier studies of Tzipori et a/. 36 However, when both strains were simultaneously fed to streptomycin-treated mice, the plasmid-cured derivative was usually unable to co-colonize the intestinal tract, suggesting that the plasmid may encode factors important in establishing colonization . Outer membrane extracts prepared from EHEC 0157 :H7 strains blocked the adherence of homologous bacteria to HEp-2 cell monolayers ." Binding inhibition was specific, since membrane extracts prepared from commensal E. co/i, purified H7 flagellin or 0157 lipopolysaccharide did not interfere with adherence . Antisera raised to a 94 kDa outer membrane protein in the extracts effectively blocked EHEC, but not EPEC, binding and actin polymerization . These data suggest that EHEC outer membrane proteins may serve as adhesins and are antigenically distinct from outer membrane proteins expressed by EPEC . The receptor(s) which EPEC and EHEC utilize to bind susceptible mammalian cells has not been characterized . Using ELISA and thin-layer chromatography techniques, Jagannatha et a/. showed that localized adherent EPEC, but not diffuse adherent EPEC, bound to a series of purified glycolipids ." The disaccharide GaINAc/31-4Gal appeared to be the common epitope necessary for binding . However, purified oligosaccharides did not block EPEC binding to HeLa cells . Although the precise glycolipids or glycoproteins necessary to mediate localized adherence remain to be identified, these data suggest that localized adherent EPEC may be members of a growing family of pathogens which use the GaINAcJf1-4Gal sequence of glycolipids as cellular receptors ." -43

Mechanisms of attaching and effacing lesion formation As was stated above, infection with EPEC is associated with the development of A/E lesions . These lesions are characterized by loss of intestinal epithelial cell microvilli and the close association (20 nm from the cell surface) ; (ii) intimate adherence, involving formation of attaching and effacing lesions (bacteria