Enteropathogenic Escherichia coli type III effectors EspG and EspG2 disrupt the microtubule network of intestinal epithelial cells

Enteropathogenic Escherichia coli infection of intestinal epithelial cells leads to localized depletion of the microtubule cytoskeleton, an effect that is dependent on delivery of type III translocated effector proteins EspG and Orf3 (designated EspG2) to the site of depletion. Microtubule depletion involved disruption rather than displacement of microtubules.     

Type III Secretion-Dependent Hemolytic Activity of Enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) was found to exhibit a type III secretion-dependent, contact-mediated, hemolytic activity requiring the EspA, EspB, and EspD secreted proteins. EspB and EspD display homology to pore-forming molecules. Our data suggest a mechanism to explain the requirement for all three Esp proteins in the transfer of EPEC proteins, such as Tir, into target cells.     

Enteropathogenic Escherichia coli Inhibits Phagocytosis

Enteropathogenic Escherichia coli (EPEC) interacts with intestinal epithelial cells, activating host signaling pathways leading to cytoskeletal rearrangements and ultimately diarrhea. In this study, we demonstrate that EPEC interacts with the macrophage-like cell line J774A.1 to inhibit phagocytosis by these cells. Antiphagocytic activity was also observed in cultured RAW macrophage-like cells upon EPEC infection. The EPEC antiphagocytic phenotype was dependent on the type III secretion pathway of EPEC and its secreted proteins, including EspA, EspB, and EspD. Intimin and Tir mutants displayed intermediate antiphagocytic activity, suggesting that intimate attachment mediated by intimin-Tir binding may also play a role in antiphagocytosis. Tyrosine dephosphorylation of several host proteins was observed following infection with secretion-competent EPEC but not with secretion-deficient mutants. Dephosphorylation was detectable 120 min after infection with EPEC, directly correlating with the onset of the antiphagocytic phenotype. Inhibition of protein tyrosine phosphatases by pervanadate treatment increased the number of intracellular wild-type EPEC organisms to levels seen with secretion-deficient mutants, suggesting that dephosphorylation events are linked to the antiphagocytic phenotype. No tyrosine phosphatase activity was detected with the EPEC-secreted proteins, suggesting that EPEC induces antiphagocytosis via a different mechanism than Yersinia species. Taken together, the present findings demonstrate a novel function for EPEC-secreted proteins in triggering macrophage protein tyrosine dephosphorylation and inhibition of phagocytosis.     

Enteropathogenic and enterohemorrhagic Escherichia coli type III secretion effector EspV induces radical morphological changes in eukaryotic cells

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic Escherichia coli (EHEC) are important human pathogens that rely on translocation of type III secretion system (T3SS) effectors for subversion of signal transduction pathways and colonization of the mammalian gut mucosa. While a core set of effectors is conserved between EPEC and EHEC strains, a growing number of accessory effectors that were found at various frequencies in clinical and environmental isolates have been recently identified. Recent genome projects identified espV as a pseudogene in EHEC but a putative functional gene in EPEC strains E110019 and E22 and the closely related mouse pathogen Citrobacter rodentium. The aim of this study was to determine the distribution of espV among clinical EPEC and EHEC strains and to investigate its function and role in pathogenesis. espV was found in 16% of the tested strains. While deletion of espV from C. rodentium did not affect colonization dynamics or fitness in mixed infections, expression of EspV in mammalian cells led to drastic morphological alterations, which were characterized by nuclear condensation, cell rounding, and formation of dendrite-like projections. Expression of EspV in yeast resulted in a dramatic increase in cell size and irreversible growth arrest. Although the role of EspV in infection and its target host cell protein(s) require further investigation, the data point to a novel mechanism by which the T3SS subverts cell signaling.     

Enteropathogenic Escherichia coli in Psittaciformes

A total of 103 Escherichia coli isolates from psittaciform birds were examined for the presence of genes coding for shigatoxin 1 (Stx1), shigatoxin 2 (Stx2) and for intimin (eae), using the polymerase chain reaction (PCR). Sixty-eight E. coli strains were isolated from necropsy cases and faecal samples, the other 35 were from 205 cloacal swabs from Psittaciformes with various conditions. All isolates were tested for enterohaemorrhagic E. coli-haemolysin (HlyEHEC), some also for Stx production, but there was no geno-typic or phenotypic evidence of Stx in any of them. Seven isolates, six from birds with diarrhoea, harboured the eae gene, three of them belonging to the O110:H6 serotype, one each to serotypes O153:H10, O131:H-, O63:H6 and Osp:H6. These seven eae-positive strains were negative for shigatoxin and HlyEHEC, and the hlyEHEC gene was not detectable by PCR. However, a PCR amplifying the enteropathogenic E. coli (EPEC)-specific bundle-forming pili structural gene bfpA detected four bfpA positive strains (three of serotype O110:H6, one O131:H-) among the seven eae positive strains, which classifies them as EPEC. Our findings suggest that shigatoxin-producing E. coli are uncommon, but that EPEC should be considered as potential pathogens in psittaciform birds, which may be a source of human EPEC infections.     

EspG, a novel type III system-secreted protein from enteropathogenic Escherichia coli with similarities to VirA of Shigella flexneri

The function of the rorf2 gene located on the locus of enterocyte effacement (LEE) pathogenicity island of enteropathogenic Escherichia coli (EPEC) has not been described. We report that rorf2 encodes a novel protein, named EspG, which is secreted by the type III secretory system and which is translocated into host epithelial cells. EspG is homologous with Shigella flexneri protein VirA, and the cloned espG (rorf2) gene can rescue invasion in a Shigella virA mutant, indicating that these proteins are functionally equivalent in Shigella. An EPEC espG mutant had no apparent defects in in vitro assays of virulence phenotypes, but a rabbit diarrheagenic E. coli strain carrying a mutant espG showed diminished intestinal colonization and yet diarrheal attack rates similar to those of the wild type. A second EspG homolog, Orf3, is encoded on the EspC pathogenicity islet. The cloned orf3 gene could also rescue invasion in a Shigella virA mutant, but an EPEC espG orf3 double mutant was not diminished in any tested in vitro assays for EPEC virulence factors. Our results indicate that EspG plays an accessory but as yet undefined role in EPEC virulence that may involve intestinal colonization.     

Enteropathogenic Escherichia coli decreases the transepithelial electrical resistance of polarized epithelial monolayers.

The mechanisms whereby enteropathogenic Escherichia coli (EPEC) causes diarrhea remain undefined. We found that EPEC caused a decrease in transepithelial electrical resistance across polarized monolayers of Caco-2 and MDCK epithelial cells. This occurred approximately 6 to 10 h after bacterial addition and was reversible if the monolayers were treated with tetracycline or gentamicin. Although significant alterations in host actin occurred beneath adherent EPEC, actin filaments supporting tight junctions were not noticeably affected in the epithelial cells, nor was the distribution of ZO-1, a tight junction protein. Despite the decrease in transepithelial electrical resistance, EPEC did not cause an increase in [3H]inulin penetration across MDCK monolayers. Unlike in the parental strain, mutations in any loci involved in adherence or formation of attaching and effacing lesions were unable to cause a decrease in transepithelial resistance. These data indicate that EPEC causes a decrease in transepithelial electrical resistance by disrupting a transcellular (intracellular) pathway rather than by disrupting intercellular tight junctions (paracellular) and that these disruptions occur only when attaching and effacing lesions are formed.     

Enteropathogenic Escherichia coli markedly decreases the resting membrane potential of Caco-2 and HeLa human epithelial cells.

It is presumed, but not proven, that enteropathogenic Escherichia coli (EPEC) causes secretory diarrhea by altering ion transport in enterocytes. In this study we used the whole-cell, current clamp variant of the patch clamp technique to demonstrate that EPEC infection of HeLa and Caco-2 human epithelial cells reduces cell resting membrane potential. The observed reduction of resting membrane potential in HeLa cells results from EPEC-mediated signal transduction to the host cell but is not dependent upon EPEC-mediated elevation of levels of intracellular free calcium. These findings indicate that EPEC can directly alter the relative distribution of ions across epithelial host cell membranes. This may be relevant to the etiology of diarrhea caused by EPEC infection.     

Enteropathogenic Escherichia coli infection: history and clinical aspects

Diarrhoeagenic Escherichia coli remains an important cause of diarrhoeal disease worldwide. In terms of global public health, enteropathogenic E. coli (EPEC) and enterotoxigenic E. coli are the most important. However, enterohaemorrhagic E. coli has emerged as a cause of disease in developed countries in recent years, and a number of large outbreaks have been reported. Therefore, the importance of research into diarrhoeagenic E. coli remains an important issue. EPEC is the most widespread of the diarrhoeagenic E. coli and provides a good virulence model for other E. coli infections, as well as other pathogenic bacteria. Although the virulence mechanisms of E. coli are now better understood, there remains much to be learned before effective treatments can be developed. Type III secretion mechanisms, the locus of enterocyte effacement and various toxins are all involved in the pathogenesis of the various diarrhoeagenic E. coli and may provide targets for future therapies. This review aims to provide an update on the worldwide problem of diarrhoeagenic E. coli by focusing on EPEC, and describes the history of the organism, its incidence and the clinical aspects of infection.     

Enteropathogenic Escherichia coli Serotypes and Endemic Diarrhea in Infants

Enteropathogenic Escherichia coli serotypes were searched for in feces of 550 children with endemic diarrhea and in 129 controls, in São Paulo, in 1978 and 1979; serotypes O111ab:H⁻, O111ab:H₂, and O119:H6 were significantly associated with diarrhea in children 0 to 5 months old and were the most frequent agents of diarrhea in this age group as compared with enterotoxigenic and enteroinvasive E. coli, Salmonella sp., Shigella sp., and Yersinia enterocolitica. It is concluded that various enteropathogenic E. coli serotypes may be agents of endemic infantile diarrhea.     

Enteropathogenic Escherichia coli infection triggers host phospholipid metabolism perturbations

Enteropathogenic Escherichia coli (EPEC) specifically recognizes phosphatidylethanolamine (PE) on the outer leaflet of host epithelial cells. EPEC also induces apoptosis in epithelial cells, which results in increased levels of outer leaflet PE and increased bacterial binding. Consequently, it is of interest to investigate whether EPEC infection perturbs host cell phospholipid metabolism and whether the changes play a role in the apoptotic signaling. Our findings indicate that EPEC infection results in a significant increase in the epithelial cell PE level and a corresponding decrease in the phosphatidylcholine (PC) level. PE synthesis via both the de novo pathway and the serine decarboxylation pathway was enhanced, and de novo synthesis of phosphatidylcholine via CDP-choline was reduced. The changes were transitory, and the maximum change was noted after 4 to 5 h of infection. Addition of exogenous PC or CDP-choline to epithelial cells prior to infection abrogated EPEC-induced apoptosis, suggesting that EPEC infection inhibits the CTP-phosphocholine cytidylyltransferase step in PC synthesis, which is reportedly inhibited during nonmicrobially induced apoptosis. On the other hand, incorporation of exogenous PE by the host cells enhanced EPEC-induced apoptosis and necrosis without increasing bacterial adhesion. This is the first report that pathogen-induced apoptosis is associated with significant changes in PE and PC metabolism, and the results suggest that EPEC adhesion to a host membrane phospholipid plays a role in disruption of host phospholipid metabolism.     

Role for CD2AP and Other Endocytosis-Associated Proteins in Enteropathogenic Escherichia coli Pedestal Formation

Enteropathogenic Escherichia coli (EPEC) strains are extracellular pathogens that generate actin-rich structures (pedestals) beneath the adherent bacteria as part of their virulence strategy. Pedestals are hallmarks of EPEC infections, and their efficient formation in vitro routinely requires phosphorylation of the EPEC effector protein Tir at tyrosine 474 (Y474). This phosphorylation results in the recruitment and direct attachment of the host adaptor protein Nck to Tir at Y474, which is utilized for actin nucleation through a downstream N-WASP-Arp2/3-based mechanism. Recently, the endocytic protein clathrin was demonstrated to be involved in EPEC pedestal formation. Here we examine the organization of clathrin in pedestals and report that CD2AP, an endocytosis-associated and cortactin-binding protein, is a novel and important component of EPEC pedestal formation that also utilizes Y474 phosphorylation of EPEC Tir. We also demonstrate the successive recruitment of Nck and then clathrin prior to actin polymerization at pedestals during the Nck-dependent pathway of pedestal formation. This study further demonstrates that endocytic proteins are key components of EPEC pedestals and suggests a novel endocytosis subversion strategy employed by these extracellular bacteria.The extracellular bacterial pathogen enteropathogenic Escherichia coli (EPEC) causes serious diarrheal disease in humans and is a prevalent microbe involved in childhood mortality in the developing world. This microbe is part of a larger family of bacteria called the attaching and effacing (A/E) pathogens that also includes the human-specific pathogen enterohemorrhagic E. coli (EHEC) and the murine disease-causing bacterium Citrobacter rodentium. These bacteria attach to intestinal epithelial cells and use a type III secretion system to directly deliver effector proteins from the bacterial cytosol into the cytoplasm of host cells. Among other functions, these effectors harness the host cell''s cytoskeleton (4) to generate actin-rich pedestals that are hallmarks of virulence for this class of pathogens (14). One of these effectors, the translocated intimin receptor (Tir), is key to pedestal formation. Following translocation into the host cell, Tir becomes embedded in the host cell plasma membrane, where its extracellular domain acts to firmly anchor the pathogen to the epithelial cell. In cultured cells, the intracellular cytoplasmic domain of EPEC Tir can become phosphorylated at tyrosine 474 (Y474) (6), where it recruits the adaptor protein Nck (7). These events all occur prior to actin filament polymerization beneath the attached bacteria via an N-WASP- and Arp2/3-based mechanism (7, 11). Although this is the prominent strategy used by EPEC to recruit actin to pedestals, a Y474-independent strategy also exists, but it occurs at a much lower frequency. During such instances, EPEC Tir becomes phosphorylated at Y454 and actin recruitment is independent of Nck (1).Previous work highlighted a role for clathrin during some bacterial infections (19, 20). Although the role of clathrin during enteropathogenic E. coli infections was not investigated until recently (20), the finding of clathrin at the tips of EPEC pedestals, coupled with the discovery of dynamin-2, another protein known to be involved in endocytosis, associated within the actin stalk of EPEC pedestals (18), suggests a possible role for additional endocytosis-associated proteins and indicates that a unique mechanism is employed by EPEC to remain extracellular despite the presence of these endocytic components. Other proteins, including the actin-associated protein cortactin, are also prominent at these structures. Cortactin is found throughout EPEC pedestals as well as pedestals formed by other attaching and effacing pathogens (2, 3). Thus, in order to further examine other endocytosis-associated proteins during EPEC pedestal formation, we opted to immunolocalize the endocytosis-related protein CD2AP (CD-2-associated protein) during these infections. CD2AP is a clathrin-associated endocytosis protein that directly associates with cortactin in other systems (12, 17).We discovered that CD2AP is present at EPEC pedestals and is a crucial component for their formation. Through the use of various host cell modification strategies, we subsequently explored the recruitment of the endocytosis-associated proteins at EPEC pedestals and found that during Nck-dependent pedestal formation, EPEC sequentially recruits Nck, clathrin, cortactin, and then CD2AP at the pedestal tip prior to the actin filament polymerization machinery at these sites.     

Evidence of Pathogenic Subgroups among Atypical Enteropathogenic Escherichia coli Strains

We describe the characterization of 126 atypical enteropathogenic Escherichia coli (aEPEC) isolates from 1,749 Brazilian children. Classic aEPEC strains were more frequently found in children with diarrhea than in controls (P < 0.001), showing their importance as acute diarrhea agents in our country. Only aEPEC strains carrying either the ehxA or paa gene were significantly associated with diarrhea.Enteropathogenic Escherichia coli (EPEC), one of the six E. coli diarrheagenic pathotypes, produces an adherence factor chromosomally encoded by the eae (EPEC attaching and effacing) gene located within the locus for enterocyte effacement (LEE) pathogenicity island (15, 16, 18).“Typical” EPEC strains contain, in addition to eae, the EPEC adherence factor (EAF) plasmid (4), which encodes the bundle-forming pili that mediate localized adherence to epithelial cells (9, 26). EPEC strains lacking the EAF plasmid have been designated “atypical” EPEC (aEPEC) (12). Whereas typical EPEC strains express only the virulence factors encoded by the LEE region and the EAF plasmid, aEPEC strains have additional virulence properties (11, 32).Recently Afset et al. (2) described several virulence genes associated with diarrhea in aEPEC isolates from Norwegian children. In their study, genes belonging to the pathogenicity island OI-122 (efa1/lifA, nleB, nleE, and sen), present in the enterohemorrhagic E. coli (EHEC) reference strain EDL933 (17), and the gene for long polar fimbriae (lpfA) (10), found in EHEC O113 strains, were particularly frequent. Other genes, such as the porcine A/E-associated gene (paa) (5) and the EHEC hemolysin gene (ehxA) (27), were also found to be associated with diarrheal disease.Other E. coli adhesion factors recently described include the protein ToxB, required for full adherence expression in EHEC O157:H7 (30); Iha, an adherence-conferring protein similar to Vibrio cholerae IrgA (29); Saa, an autoagglutinating adhesin identified in LEE-negative strains (22); and Spf, a sorbitol-fermenting EHEC O157 fimbria (8). In addition, we recently identified a diffuse adherence (lda) locus in an aEPEC strain of the O26 serogroup that codes for adherence to HEp-2 cells (25).In this report, we describe the prevalence and virulence profile of aEPEC strains isolated from diarrhea patients and control subjects in several cities of Brazil from 1999 through 2004.Stool specimens from 1,102 children under 2 years of age with diarrhea, presenting to the emergency room of public hospitals in seven cities representing different regions of Brazil, and 647 randomly selected children without any gastrointestinal symptoms from the same hospitals were studied. All specimens were investigated for the presence of enteric pathogens, such as diarrheagenic E. coli, Shigella species, Salmonella species, Yersinia enterocolitica, Campylobacter species, and rotavirus (24).Atypical EPEC strains were isolated, identified, and serotyped as described elsewhere (11). One isolate per subject was stored at −70°C.aEPEC strains were screened by colony blot hybridization with 16 different DNA probes representing a panel of toxin, adhesin, and OI-122 genes. Fourteen DNA probes were prepared by PCR amplification from prototype strains. The genes, primers, amplicon size, PCR conditions, and prototype strains are given in Table ​Table1.1. Two DNA probes were prepared by plasmid extraction using the method of Birnboim and Doly (7) and digestion with appropriate restriction endonucleases. The cdt probe was a 1,357-bp fragment from plasmid pCVD448 (28), and the cnf probe was a 335-bp fragment of pEOSW1 (20). The probes were then purified by gel extraction (23) and labeled with [α-³²P]dCTP, and colony hybridization assays were performed as described elsewhere (24).

TABLE 1.

PCR primers and conditions used in this study and sizes of PCR amplicons