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Human intestinal organoid cultures established from crypt-derived stem cells truly revolutionized our approach to study intestinal epithelial physiology and pathologies as they can be propagated indefinitely and preserve the genetic signature of the donor and the gut segment specificity in culture. Here we describe human stem cell-derived colonoid monolayers as a reliable and reproducible model to study Shiga toxin-producing Escherichia coli (STEC) infection and STEC-caused pathologies of the whole colonic epithelium comprising a mixture of colonocytes, goblet, enteroendocrine, and other rare cells present in human colonic epithelial tissue.

Attaching and effacing Escherichia coli (AEEC) strains are a genomically diverse group of diarrheagenic E. coli strains that are characterized by the presence of the locus of enterocyte effacement (LEE) genomic island, which encodes a type III secretion system that is essential to virulence. AEEC strains can be further classified as either enterohemorrhagic E. coli (EHEC), typical enteropathogenic E. coli (EPEC), or atypical EPEC, depending on the presence or absence of the Shiga toxin genes or bundle-forming pilus (BFP) genes. Recent AEEC genomic studies have focused on the diversity of the core genome, and less is known regarding the genetic diversity and relatedness of AEEC plasmids. Comparative genomic analyses in this study demonstrated genetic similarity among AEEC plasmid genes involved in plasmid replication conjugative transfer and maintenance, while the remainder of the plasmids had sequence variability. Investigation of the EPEC adherence factor (EAF) plasmids, which carry the BFP genes, demonstrated significant plasmid diversity even among isolates within the same phylogenomic lineage, suggesting that these EAF-like plasmids have undergone genetic modifications or have been lost and acquired multiple times. Global transcriptional analyses of the EPEC prototype isolate E2348/69 and two EAF plasmid mutants of this isolate demonstrated that the plasmid genes influence the expression of a number of chromosomal genes in addition to the LEE. This suggests that the genetic diversity of the EAF plasmids could contribute to differences in the global virulence regulons of EPEC isolates.

(See the editorial commentary by Steiner on pages 1860–2.) Enteroaggregative Escherichia coli (EAEC) is a pathotype of diarrheagenic Escherichia coli that is a cause of acute and persistent diarrhea in many settings [1–7]. EAEC strains express a heterogeneous array of putative virulence factors [8–12] encoded on the bacterial chromosome or on the EAEC-specific pAA plasmid. EAEC strains often harbor a transcriptional activator of the AraC/XylS class, called “AggR” [13], which controls genes on both the plasmid and the chromosome. Among the genes under AggR control include those that encode the aggregative adherence fimbriae (AAF), of which at least 4 variants exist [14–18]. AAF adhesins have been shown to be essential for EAEC adherence to human intestinal explants and to elicit both cytokine release and opening of epithelial tight junctions in a polarized epithelial model [19, 20]. EAEC strains also often harbor a variable number of serine protease autotransporters of Enterobacteriaceae (SPATEs) that are implicated in immune evasion, mucosal damage, secretogenicity, and colonization [21]. In 2011, an outbreak of foodborne hemorrhagic colitis originated in Germany, spreading to other European countries. Over 4000 individuals were affected, including primary and secondary cases [22]. Hemolytic uremic syndrome (HUS) developed in approximately 22% of the cases [22], and 54 people died [23, 24]. The implicated pathogen was an EAEC strain of the rare serotype O104:H4 [23], which was lysogenized with an Stx2a-converting phage. Genomic analysis [25] demonstrated that the outbreak strain contained the genes required to produce the AAF/I variant and 3 SPATEs (Pic, SigA, and SepA). Although much is known about the pathogenesis of serotype O157:H7 Shiga toxin (Stx)–producing E. coli, it is unclear how an EAEC strain would be able to elicit severe hemorrhagic colitis and HUS, even when harboring an Stx-encoding gene. In this study, we tested the hypothesis that the plasmid-borne virulence factors of EAEC contributed to the high pathogenicity of the German outbreak strain by promoting strong adherence to the epithelium and/or by opening epithelial tight junctions.

Enterohemorrhagic Escherichia coli (EHEC) is a food-borne pathogen that can cause severe health complications and utilizes a much lower infectious dose than other E. coli pathotypes. Despite having an intact ure locus, ureDABCEFG , the majority of EHEC strains are phenotypically urease negative under tested conditions. Urease activity potentially assists with survival fitness by enhancing acid tolerance during passage through the stomach or by aiding with colonization in either human or animal reservoirs. Previously, in the EHEC O157:H7 Sakai strain, a point mutation in ureD , encoding a urease chaperone protein, was identified, resulting in a substitution of an amber stop codon for glutamine. This single nucleotide polymorphism (SNP) is observed in the majority of EHEC O157:H7 isolates and correlates with a negative urease phenotype in vitro . We demonstrate that the lack of urease activity in vitro is not solely due to the amber codon in ureD. Our analysis has identified two additional SNPs in ureD affecting amino acid positions 38 and 205, in both cases determining whether the encoded amino acid is leucine or proline. Phylogenetic analysis based on Ure protein sequences from a variety of urease-encoding bacteria demonstrates that the proline at position 38 is highly conserved among Gram-negative bacteria. Experiments reveal that the L38P substitution enhances urease enzyme activity; however, the L205P substitution does not. Multilocus sequence typing analysis for a variety of Shiga toxin-producing E. coli isolates combined with the ureD sequence reveals that except for a subset of the O157:H7 strains, neither the in vitro urease-positive phenotype nor the ureD sequence is phylogenetically restricted.

One of the characteristic manifestations of Shiga-toxin-producing Escherichia coli (E. coli) infection in humans, including EHEC and Enteroaggregative E. coli O104:H4, is watery diarrhea. However, neither Shiga toxin nor numerous components of the type-3 secretion system have been found to independently elicit fluid secretion. We used the adult stem-cell-derived human colonoid monolayers (HCM) to test whether EHEC-secreted extracellular serine protease P (EspP), a member of the serine protease family broadly expressed by diarrheagenic E. coli can act as an enterotoxin. We applied the Ussing chamber/voltage clamp technique to determine whether EspP stimulates electrogenic ion transport indicated by a change in short-circuit current (Isc). EspP stimulates Isc in HCM. The EspP-stimulated Isc does not require protease activity, is not cystic fibrosis transmembrane conductance regulator (CFTR)-mediated, but is partially Ca2+-dependent. EspP neutralization with a specific antibody reduces its potency in stimulating Isc. Serine Protease A, secreted by Enteroaggregative E. coli, also stimulates Isc in HCM, but this current is CFTR-dependent. In conclusion, EspP stimulates colonic CFTR-independent active ion transport and may be involved in the pathophysiology of EHEC diarrhea. Serine protease toxins from E. coli pathogens appear to serve as enterotoxins, potentially significantly contributing to watery diarrhea.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 responds to the host-produced epinephrine and norepinephrine, and bacterially produced autoinducer 3 (AI-3), through two-component systems. Further integration of multiple regulatory signaling networks, involving regulators such as the LysR-type transcriptional regulator (LTTR) QseA, promotes effective regulation of virulence factors. These include the production of flagella, a phage-encoded Shiga toxin, and genes within the locus of enterocyte effacement (LEE) responsible for attaching and effacing (AE) lesion formation. Here, we describe a new member of this signaling cascade, an LTTR heretofore renamed QseD (quorum-sensing E. coli regulator D). QseD is present in all enterobacteria but exists almost exclusively in O157:H7 isolates as a helix-turn-helix (HTH) truncated isoform. This “short” isoform (sQseD) is still able to regulate gene expression through a different mechanism than the full-length K-12 E. coli “long” QseD isoform (lQseD). The EHEC Δ qseD mutant exhibits increased expression of all LEE operons and deregulation of AE lesion formation. The loss of qseD in EHEC does not affect motility, but the K-12 Δ qseD mutant is hypermotile. While the lQseD directly binds to the ler promoter, encoding the LEE master regulator, to repress LEE transcription, the sQseD isoform does not. LTTRs bind to DNA as tetramers, and these data suggest that sQseD regulates ler by forming heterotetramers with another LTTR. The LTTRs known to regulate LEE transcription, QseA and LrhA, do not interact with sQseD, suggesting that sQseD acts as a dominant-negative partner with a yet-unidentified LTTR.

Pathogens and eucaryotic cells are active partners during the process of pathogenicity. To gain access to enterocytes and to cross the epithelial membrane, many enterovirulent microorganisms interact with the brush border membrane-associated components as receptors. Recent reports provide evidence that intestinal cell differentiation plays a role in microbial pathogenesis. Human enteropathogenic Escherichia coli (EPEC) develop their pathogenicity upon infecting enterocytes. To determine if intestinal epithelial cell differentiation influences EPEC pathogenicity, we examined the infection of human intestinal epithelial cells by JPN 15 (pMAR7) [EAF+ eae+] EPEC strain as a function of the cell differentiation. The human embryonic intestinal INT407 cells, the human colonic T84 cells, the human undifferentiated HT-29 cells (HT-29 Std) and two enterocytic cell lines, HT-29 glc−/+ and Caco-2 cells, were used as cellular models. Cells were infected apically with the EPEC strain and the cell-association and cell-entry were examined by quantitative determination using metabolically radiolabeled bacteria, as well as by light, scanning and tranmission electron microscopy. [EAF+ eae+] EPEC bacteria efficiently colonized the cultured human intestinal cells. Diffuse bacterial adhesion occurred to undifferentiated HT-29 Std and INT407 cells, whereas characteristic EPEC cell clusters were observed on fully differentiated enterocytic HT-29 glc−/+ cells and on colonic crypt T84 cells. As shown using the Caco-2 cell line, which spontaneously differentiates in culture, the formation of EPEC clusters increased as a function of the epithelial cell differentiation. In contrast, efficient cell-entry of [EAF+ eae+] EPEC bacteria occured in recently differentiated Caco-2 cells and decreased when the cells were fully differentiated. Our results provide evidence that the intestinal cell differentiation could play a dual role in EPEC pathogenesis: it up-regulates intestinal cell colonization and down-regulates intestinal cell invasion.

The human pathogen Shiga-toxigenic Escherichia coli (STEC) O157:H7 contains a ycbQRST fimbrial-like operon, which shares significant homology to the family of F17 fimbrial biogenesis genes f17ADCG found in enterotoxigenic E. coli. We report that growth of STEC O157:H7 strain EDL933 in minimal Minca medium at 37°C and during adherence to epithelial cells led to the production of fine peritrichous fimbriae, which were found to be composed of a major subunit of 18 kDa whose N-terminal amino acid sequence matched the predicted protein product of the ycbQ gene; and showed significant homology to the F17a-A fimbrin. Similar to the F17 fimbriae, the purified STEC fimbriae and the recombinant YcbQ protein fused to a His peptide tag bound laminin, but not fibronectin or collagen. Thus, we propose the name E. coli YcbQ laminin-binding fimbriae (ELF) to designate the fimbriae encoded by the ycbQRST operon. The role of ELF as an adherence factor of STEC to cultured epithelial cells was investigated. We provide compelling evidence demonstrating that ELF contributes to adherence of STEC to human intestinal epithelial cells and to cow and pig gut tissue in vitro. Deletion in the fimbrin subunit gene elfA (or ycbQ) in STEC strain EDL933 led to an isogenic strain, which showed significant reduction (60%) in adherence to HEp-2 cells in comparison with the parental strain. In addition, antibodies against the purified ELF also partially blocked adherence of two STEC O157:H7 strains. These observations suggest that ELF functions as an accessory adherence factor that, along with other known redundant adhesins, contributes to the overall adhesive properties of STEC O157:H7 providing these organisms with ecological advantages to survive in different hosts and in the environment.

Enterotoxigenic Escherichia coli (ETEC) strains produce a type IV pilus named Longus. We identified a 16-gene cluster involved in the biosynthesis of Longus that has 57 to 95% identity at the protein level to CFA/III, another type IV pilus of ETEC. Alleles of the Longus structural subunit gene lngA demonstrate a diversity of 12 to 19% at the protein level with strong positive selection for point replacements and horizontal transfer.

Attaching and effacing (A/E) organisms, such as rabbit enteropathogenic Escherichia coli (EPEC), human EPEC or enterohemorrhagic E. coli (EHEC) share attaching and effacing phenotype and LEE pathogenicity island responsible for A/E. The present study was undertaken to investigate the impact of the LuxS quorum sensing (QS) signaling system in vitro and in vivo pathogenicity of A/E organisms using rabbit EPEC (rEPEC) strain E22 (O103:H2). Analysis of the bioluminescence indicated abolished production of the QS signal AI-2 by luxS mutant (E22DeltaluxS). Strain E22Deltalux also exhibited impaired expression of several normally secreted proteins and reduced adherence to cultured HeLa cells. Complementation of the intact luxS gene to E22DeltaluxS restored secreted protein expression comparable to the WT type but not adherence to HeLa cells. In experimentally infected rabbits, the isogenic luxS mutant induced clinical illness and intimate adherence to the intestinal mucosa, albeit to a less extent, comparable to that seen with the parent virulent strain. It is worth noting that reduced fecal bacterial shedding, mucosal adherence and improved cumulative weight gain were seen for the mutant strain complemented with luxS when compared to the WT. It appears that the luxS gene is not essential for in vivo pathogenicity by rEPEC where exogenous QS signals are present in the gut. The impact of AI-2 provided by multicopy plasmid on bacterial virulence is discussed.

Members of the genus Shigella are phenotypically similar to Escherichia coli and, with the exception of Shigella boydii serotype 13, would be considered the same species by DNA-DNA hybridization analysis and whole-genome sequence analysis. The dynamic nature of the Shiga toxin-converting phages has implications for diagnostic testing for Shiga toxin-producing E. coli (STEC). Since STEC strains can lose critical virulence genes, some researchers have proposed that multiple virulence-associated genes, as well as conserved genes, be used to diagnose infections by these bacteria. This concept would also apply to other pathotypes of E. coli, as most of them carry critical virulence genes on mobile genetic elements. A preliminary report can be issued as soon as a presumptive identification of Salmonella is obtained. In most situations, a presumptive identification is based on phenotypic traits determined by either traditional or commercial systems or by reactivity with Salmonella O grouping antisera. A confirmed identification requires both phenotypic identification and O group or serotype determination. As national surveillance systems depend on the receipt of serotype information for Salmonella strains isolated in the United States, laboratories should follow the procedures recommended by their state health departments for submitting Salmonella isolates for further characterization, including complete serotyping. The antimicrobial susceptibilities of typhoidal Salmonella strains and strains from normally sterile sites should be determined, and the strains should be forwarded to a reference or public health laboratory for complete phenotypic identification and serotyping.

In this overview, we describe the history of Shiga toxin (Stx)-producing Escherichia coli (STEC) in two phases. In phase one, between 1977 and 2011, we learned that E. coli could produce Shiga toxin and cause both hemorrhagic colitis and the hemolytic-uremic syndrome in humans and that the prototype STEC— E. coli O157:H7—adheres to and effaces intestinal epithelial cells by a mechanism similar to that of enteropathogenic E. coli . We also recognized that the genes for Stx are typically encoded on a lysogenic phage; that STEC O157:H7 harbors a large pathogenicity island that encodes the elements needed for the characteristic attaching and effacing lesion; and that the most severe cases of human disease are linked to production of Stx type 2a, not Stx type 1a. Phase two began with a large food-borne outbreak of hemorrhagic colitis and hemolytic-uremic syndrome in Germany in 2011. That outbreak was caused by a novel strain consisting of enteroaggregative E. coli O104:H4 transduced by a Stx2a-converting phage. From this outbreak we learned that any E. coli strain that can adhere tightly to the human bowel (either by a biofilm-like mechanism as in E. coli O104:H4 or by an attaching and effacing mechanism as in E. coli O157:H7) can cause severe diarrheal and systemic illness when it acquires the capacity to produce Stx2a. This overview provides the basis for the review of current information regarding these fascinating and complex pathogens.

The dysenteric Shigella spp. and enteroinvasive Escherichia coli (EIEC) have evolved from commensal E. coli by the acquisition of a virulence plasmid and inactivation of genes of the cad locus encoding lysine decarboxylase (LDC) by so-called pathoadaptive mutation. As horizontal gene transfer and recombination occurs frequently in E. coli we were interested to see if similar pathoadaptive mutations are commonly present in other intestinal pathotypes. Therefore, we examined 140 intestinal E. coli strains of various pathotypes and the ECOR collection for their ability to decarboxylate lysine, and identified 25 strains that were unable to do so. Complementation of a Shiga toxin-producing E. coli and two enteropathogenic E. coli strains, both LDC-negative, with the intact cad locus restored LDC activity and resulted in a reduction in adherence to tissue culture cells. We investigated the cad locus for possible alterations by using hybridization and PCR techniques and compared the results with the alterations reported for Shigella spp. and EIEC strains. Interestingly, the alterations of the cad genes were similar to those previously reported, pointing towards a parallel evolution of LDC silencing in different intestinal E. coli pathotypes.

The complete nucleotide sequence was determined for pMAR7, an enteropathogenic Escherichia coli (EPEC) adherence factor (EAF) plasmid that contains genes encoding a type IV attachment pilus (Bfp) and the global virulence regulator per . Prototypic EAF plasmid pMAR7 is self-transmissible, unlike the smaller EAF plasmid pB171, which has no genes encoding conjugative functions. The tra locus, a highly conserved 33-kb segment found in pMAR7, is similar to the tra (conjugation) region of the F plasmid. IS Ec13 copies flanking the pMAR7 tra region could potentially mobilize or delete the tra genes. Hybridization of 134 EPEC strains showed that a complete tra region is present only in strains of the EPEC1 clonal group. This study confirms EPEC's potential for dissemination of virulence attributes by horizontal transfer of the EAF plasmid.

The ler (LEE encoded regulator) gene product is a central regulator for the genes encoded on the locus of enterocyte effacement (LEE) pathogenicity island of attaching/effacing (A/E) pathogens, including human enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) as well as animal isolates. Although an in vivo role for Ler in bacterial virulence has not been documented, we hypothesized that a Ler deletion mutant should be attenuated for virulence but might retain immunogenicity. The goals of this study were to genetically characterize ler of a rabbit EPEC (rEPEC) strain (O103:H2), to examine the effect of ler on in vivo virulence, and to determine if intragastric inoculation of an attenuated rEPEC ler mutant was immunogenic and could protect rabbits against subsequent challenge with the wild-type virulent parent strain. The predicted ler gene product of rEPEC strain O103:H2 shares high homology (over 95% amino acid identity) with the Lers of another rEPEC strain RDEC-1 (O15:H-) and human EPEC and EHEC. A defined internal ler deletion mutant of rEPEC O103:H2 showed reduced production of secreted proteins. Although orogastric inoculation of rabbits with the virulent parent O103:H2 strain induced severe diarrhea, significant weight loss and early mortality with adherent mucosal bacteria found at sacrifice, the isogeneic ler mutant strain was well tolerated. Animals gained weight and showed no clinical signs of disease. Examination of histological sections of intestinal segments revealed the absence of mucosal bacterial adherence. This result demonstrates an essential role for Ler in in vivo pathogenicity of A/E E. coli. Single dose orogastric immunization with the rEPEC ler mutant induced serum IgG antibody to whole bacteria (but not to intimin). Immunized animals were protected against enteric infection with the WT virulent parent strain exhibiting normal weight gain, absence of diarrhea and absence of mucosally adherent bacteria at sacrifice. Such attenuated ler mutant strains may have potential for use as oral vaccines, or as vaccine vectors for delivery of foreign antigens. It remains to be determined whether such regulatory mutants can protect against infection with A/E bacteria of differing serotypes affecting different hosts.

Using the enteropathogenic Escherichia coli (EPEC) genome sequence, we found that EPEC E2348/69 has an lpfABCDE gene cluster homologous (about 60% identical at the protein level) to the Salmonella long polar fimbria (LPF) operon. To determine whether this operon is essential for adherence, the lpfABCD E2 3 genes were deleted from EPEC strain E2348/69 by allelic exchange. Analysis of the resulting EPEC ΔlpfABCD E23 strain showed no change in adherence to HeLa cells or to human intestinal biopsy cells in the in vitro organ culture (IVOC) system compared to the wild type. Sera from volunteers experimentally infected with E2348/69 showed no antibody response to the major subunit protein, LpfA. These results suggested that the lpf E23 gene cluster is not necessary for EPEC adherence and attaching/effacing (A/E) lesion formation on human biopsy samples and is not expressed during human infection. We also identified an lpf gene cluster in Citrobacter rodentium strain ICC168 ( lpf cr ). A Δ lpfA cr mutant of ICC168 retained wild-type adherence and A/E lesion-forming activity on HeLa cells. C3H/HeJ mice were infected with a wild-type C. rodentium strain and its lpfA cr isogenic mutant. Both strains were recovered at high levels in stools, and there were no significant differences between the groups both in terms of the number of CFU/organ (colon and cecum) and in terms of the amount of hyperplasia, as measured by weight. Similar results were observed in a second mouse strain, C57BL/6. These data suggest that in addition to playing no apparent role in EPEC pathogenesis, lpf cr is not required for C. rodentium virulence in either the C3H/HeJ or C57BL/6 mouse model.

The contribution of long polar fimbriae to intestinal colonization by Escherichia coli O157:H7 was evaluated in sheep, conventional pigs, and gnotobiotic piglets. E. coli O157:H7 strains with lpfA1 and lpfA2 mutated were recovered in significantly lower numbers and caused fewer attachment and effacement lesions than the parent strain.

A second region containing five genes homologous to the long polar fimbrial operon of Salmonella enterica serovar Typhimurium is located in the chromosome of enterohemorrhagic Escherichia coli (EHEC) O157:H7. A non-fimbriated E. coli K-12 strain carrying the cloned EHEC lpf (lpf2) genes expressed thin fibrillae-like structures on its surface and displayed reduced adherence to tissue culture cells. Neither mutation in the lpfA2 gene in either the parent or lpfA1 mutant strains showed an effect in adherence or in the formation of A/E lesions on HeLa cells. lpfA2 isogenic mutant strains adhere to Caco-2 cells almost as well as the wild-type at 5 h, but they were deficient in adherence at early time points. A collection of diarrheagenic E. coli strains were investigated for the presence of lpfA1 and lpfA2 and results showed that these genes are present in specific serogroups which are phylogenetically related. Our results suggest that LP fimbriae 2 may contribute to the early stages of EHEC adhesion and that genes encoding the major LP fimbrial subunits are present in a small group of EHEC and EPEC serotypes.

The distribution of EDL 933 O island 122 (OI-122) was investigated in 70 strains of Verocytotoxin-producing Escherichia coli (VTEC) of multiple serotypes that were classified into five “seropathotypes” (A through E) based on the reported occurrence of serotypes in human disease, in outbreaks, and/or in the hemolytic-uremic syndrome (HUS). Seropathotype A comprised 10 serotype O157:H7 and 3 serotype O157:NM strains. Seropathotype B (associated with outbreaks and HUS but less commonly than serotype O157:H7) comprised three strains each of serotypes O26:H11, O103:H2, O111:NM, O121:H19, and O145:NM. Seropathotype C comprised four strains each of serotypes O91:H21 and O113:H21 and eight strains of other serotypes that have been associated with sporadic HUS but not typically with outbreaks. Seropathotype D comprised 14 strains of serotypes that have been associated with diarrhea but not with outbreaks or HUS, and seropathotype E comprised animal VTEC strains of serotypes not implicated in human disease. All strains were tested for four EDL 933 OI-122 virulence genes (Z4321, Z4326, Z4332, and Z4333) by PCR. Negative PCRs were confirmed by Southern hybridization. Overall, 28 (40%) strains contained OI-122 (positive for all four virulence genes), 27 (38.6%) contained an “incomplete” OI-122 (positive for one to three genes), and 15 (21.4%) strains did not contain OI-122. The seropathotype distribution of complete OI-122 was as follows: 100% for seropathotype A, 60% for B, 36% for C, 15% for D, and 0% for E. The differences in the frequency of OI-122 between seropathotypes A, B, and C (associated with HUS) and seropathotypes D and E (not associated with HUS) and between seropathotypes A and B (associated with epidemic disease) and seropathotypes C, D, and E (not associated with epidemic disease) were highly significant ( P < 0.0001).

Stool specimens from 113 adult outpatients with diarrhea in southwestern Nigeria and 63 controls were examined for bacterial and parasitic enteric pathogens. Enterohemorrhagic Escherichia coli (EHEC) ( P < 0.02), enteroaggregative E. coli (EAEC) ( P < 0.02), and Entamoeba histolytica ( P < 0.0002) were significantly associated with diarrhea. Salmonella , Shigella , nontoxigenic Vibrio cholerae , other categories of diarrheagenic E. coli , as well as a variety of helminths were recovered more frequently from the stools of patients than from the stools of controls but did not show a significant association with disease. Multiple pathogens were recovered from 36.3% of specimens, and bloody diarrhea was commonly associated with E. histolytica and diarrheagenic E. coli infections. The majority of EHEC isolates were non-O157 strains that carried the stx 2 gene. Of the 23 EHEC-infected patients, 12 (52.2%) presented during the 10th week of the study. EHEC strains isolated within this cluster were more likely to hybridize with the enterohemolysin gene probe, to be nonmotile and sorbitol positive, and to fail to agglutinate O157 antisera. Pulsed-field gel electrophoresis demonstrated that the only strains with Xba I profiles that occurred more than once were isolated during the 10th and 11th weeks of the study, suggesting an outbreak. The study has demonstrated that E. histolytica , EHEC, and EAEC are important diarrheal pathogens within the study area and that sporadic and epidemic EHEC infections occur in developing as well as developed countries. Routine surveillance for diarrheagenic E. coli , even only at the tertiary-care level, would be useful in identifying outbreaks and assist in identifying environmental reservoirs and transmission routes.

Adherence of enterohemorrhagic Escherichia coli (EHEC) to the intestinal epithelium is essential for initiation of infection. Intimin is the only factor demonstrated to play a role in intestinal colonization by EHEC O157:H7. Other attempts to identify additional adhesion factors in vitro have been unsuccessful, suggesting that expression of these factors is under tight regulation. We sought to identify genes involved in the control of adherence of EHEC O157:H7 to cultured epithelial cells. A total of 5,000 independent transposon insertion mutants were screened for their ability to adhere to HeLa cells, and 7 mutants were isolated with a markedly enhanced adherence. The mutants adhered at levels 113 to 170% that of the wild-type strain, and analysis of the protein profiles of these mutants revealed several proteins differentially expressed under in vitro culture conditions. We determined the sequence of the differentially expressed proteins and further investigated the function of OmpA, whose expression was increased in a mutant with an insertionally inactivated tcdA gene. An isogenic ompA mutant showed reduced adherence compared to the parent strain. Disruption of the ompA gene in the tdcA mutant strain abolished the hyperadherent phenotype, and anti-OmpA serum inhibited adhesion of wild-type and tdcA mutant strains to HeLa cells. Enhanced adhesion mediated by OmpA was also observed with Caco-2 cells, and anti-OmpA serum blocked adherence to HeLa cells of other EHEC O157:H7 strains. Our results indicate that multiple elements control adherence and OmpA acts as an adhesin in EHEC O157:H7.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is recognized as a significant enteric pathogen that has been implicated in numerous outbreaks worldwide (reviewed in reference 24). This organism colonizes the intestine and can cause bloody or nonbloody diarrhea and hemolytic uremic syndrome. A potent cytotoxin, Shiga toxin (Stx), is the best-characterized virulence factor, but many aspects of the pathogenesis of the disease associated with E. coli O157:H7 are poorly characterized. In particular, the mechanisms underlying the adherence of EHEC to intestinal epithelial cells are not well understood (24, 27). Colonization of the gastrointestinal tract, which is presumably mediated by specific adherence factors, is a key aspect of enteric infection caused by EHEC. Several potential virulence determinants of EHEC have been described, but the only adherence factor that has been demonstrated to play a role in intestinal colonization in vivo in an animal model is the outer membrane protein intimin (8, 22, 43). Most EHEC and all enteropathogenic E. coli (EPEC) strains produce this adhesin (17). Intimin, encoded by the eae gene, is located within the locus for enterocyte effacement (LEE) pathogenicity island, which is required for the classic attaching and effacing intestinal lesion produced by these organisms (14, 15, 21). The presence of a second adherence factor has been described in EPEC but not in EHEC strains. The type IV bundle-forming pilus (BFP) encoded by the plasmid of EPEC strains is involved in bacterium-to-bacterium adherence in the localized adherence pattern (4, 12) and potentially in direct interaction with the host epithelial cells (41, 42). Neither BFP nor analogous adhesins have been described in EHEC, but the existence of intestinal adherence factors distinct from intimin is suggested by the isolation of human EHEC strains of serotypes other than O157:H7 that lack the eae gene but are still associated with bloody diarrhea or hemolytic uremic syndrome (10). Several research groups have explored this hypothesis and proposed potential novel adhesin factors. Tarr et al. (37) identified Iha (Vibrio cholerae IrgA-homologue adhesin) in E. coli O157:H7. This protein was associated with adherence to HeLa cells while expressed in a nonfimbriated E. coli strain, but no difference in adherence was observed with an iha mutant of O157:H7. Nicholls et al. (25) characterized a chromosomal genetic locus termed efa1 (EHEC factor for adherence) in an EHEC strain serotype O111:H− and observed that this locus is associated with high levels of adherence to cultured Chinese hamster ovary (CHO) cells. They demonstrated that the efa1 locus was necessary for the in vitro adhesion to CHO cells and that the efa1 isogenic mutant strain lost its ability to adhere and also was defective in its hemagglutination and autoaggregation phenotypes. Tatsuno et al. (39) performed a transposon mutagenesis in the EHEC O157:H7 strain (O157Sakai), and the insertion mutants were screened for their ability to adhere to Caco-2 cells. Almost half of the insertion mutants were found within the LEE pathogenicity island, while the other half were mapped to open reading frames (ORFs) with unknown functions or with functions not directly associated with adherence. Their results suggested that EHEC might contain additional adherence-associated loci which are not contained within the LEE pathogenicity island. Recently, Brunder et al. (6) characterized a gene cluster in the large plasmid of a sorbitol-fermenting EHEC O157:H− strain which is required for the expression of fimbriae with homology to the P fimbriae of uropathogenic E. coli. The Sfp (for sorbitol-fermenting EHEC O157 fimbriae, plasmid encoded) fimbriae mediate mannose-resistant hemagglutination, but this sfp gene cluster is restricted to sorbitol-fermenting EHEC O157:H− strains and is absent in other EHEC serotypes, including O157:H7. In this work, we characterized a chromosomal fimbrial operon of E. coli O157:H7. Sequence analysis indicated that this operon showed high similarity to the long polar (LP) fimbria (lpf) operon of Salmonella enterica serovar Typhimurium and to a lesser degree to other well-characterized fimbrial operons. Introduction of the EHEC lpf operon into a nonfimbriated E. coli K-12 strain resulted in the expression of fimbriae and increased adhesion to tissue culture cells. We also provide evidence suggesting that LP fimbriae participate in the adherence of E. coli O157:H7 to eukaryotic cells and play a role in microcolony formation.

Escherichia coli O157:H7 causes diarrhoea, haemorrhagic colitis, and the haemolytic uraemic syndrome. We have identified a protein of previously unknown function encoded on the pO157 virulence plasmid of E. coli O157:H7, which is the first described protease that specifically cleaves C1 esterase inhibitor (C1-INH), a member of the serine protease inhibitor family. The protein, named StcE for secreted protease of C1 esterase inhibitor from EHEC (formerly Tagn), cleaves C1-INH to produce (unique) approximately 60-65 kDa fragments. StcE does not digest other serine protease inhibitors, extracellular matrix proteins or universal protease targets. We also observed that StcE causes the aggregation of cultured human T cells but not macrophage-like cells or B cells. Substitution of aspartic acid for glutamic acid at StcE position 435 within the consensus metalloprotease active site ablates its abilities to digest C1-INH and to aggregate T cells. StcE is secreted by the etp type II secretion pathway encoded on pO157, and extracellular StcE levels are positively regulated by the LEE-encoded regulator, Ler. StcE antigen and activity were detected in the faeces of a child with an E. coli O157:H7 infection, demonstrating the expression of StcE during human disease. Cleavage of C1-INH by StcE could plausibly cause localized pro-inflammatory and coagulation responses resulting in tissue damage, intestinal oedema and thrombotic abnormalities.

Enteropathogenic Escherichia coli (EPEC) utilizes a type III protein secretion system to target effector molecules into the host cell leading to effacement of the intestinal mucosa. This secretion apparatus shares many structural features of the flagellar type III export system involved in flagella assembly and motility. We report here that fliC insertional mutants constructed in two wild-type EPEC strains were markedly impaired in adherence and microcolony formation on cultured cells. An E. coli K-12 strain harbouring the EPEC H6 fliC gene on a plasmid showed discrete adhering clusters on HeLa cells, albeit to less extent than the wild-type EPEC strain. Flagella purified from EPEC bound to cultured epithelial cells and antiflagella antibodies blocked adherence of several EPEC serotypes. We determined that eukaryotic cells in culture stimulate expression of flagella by motile and non-motile EPEC. Isogenic strains mutated in perA (a transcriptional activator), bfpA (a type IV pilin), luxS (a quorum-sensing autoinducer gene) and in the type III secretion genes were reduced for motility in Dulbecco's modified Eagle medium (DMEM) motility agar and produced none or few flagella when associated with epithelial cells. Growth of these mutants in preconditioned tissue culture medium restored motility and their ability to produce flagella, suggesting the influence of a signal provided by mammalian cells that triggers flagella production. This study shows for the first time that the flagella of EPEC are directly involved in the adherence of these bacteria and supports the existence of a molecular relationship between the two existing type III secretion pathways of EPEC, the EPEC adherence factor (EAF) plasmid-encoded regulator, quorum sensing and epithelial cells.

Escherichia coli are generally considered to be members of the common, indigenous microbiota of the human and other animal intestinal tracts. In fact, they are generally beneficial in several ways. Several virotypes (particular virulent strains) within the species that have the capacity for causing gastrointestinal disease in humans were discovered within the last 50 years. The virotypes have been studied particularly for their epidemiology and the mechanisms by which they are able to cause disease. The virotypes of E. coli described in this review are enterotoxigenic, enteropathogenic, enteroaggregative, enteroinvasive, diffusely-adherent, and enterohemorrhagic (EHEC). Many of the virotypes are responsible for causing widespread outbreaks of diarrheal disease, most of which are self-limiting. The exceptions are the EHEC that are featured in this review. EHEC produce shiga-like toxins and are capable of causing hemorrhagic enterocolitis and hemolytic uremic syndrome, a potentially fatal disease. # 2002 Lippincott Williams & Wilkins

The properties of 23 cell-detaching Escherichia coli strains that were isolated from stool specimens in Nigeria are described. Common properties of the strains included the presence of genes encoding α-hemolysin (100%), pyelonephritis-associated pili (100%), and cytotoxic necrotizing factor 1 (70%) as well as lactose negativity (70%) and multiple antibiotic resistance (74%). Antibiotic resistance was shown in most cases to be transferable and associated with the presence of class 1 integrons. Phenotypic properties and pulsed-field gel electrophoresis analysis demonstrated that the majority of the strains, particularly multiply resistant, lactose-negative O4:H40 strains, were closely related. Multiply-resistant cell-detaching E. coli strains may represent an important reservoir for antibiotic resistance genes.

In many Gram-negative bacteria, a key indicator of pathogenic potential is the possession of a specialized type III secretion system, which is utilized to deliver virulence effector proteins directly into the host cell cytosol. Many of the proteins secreted from such systems require small cytosolic chaperones to maintain the secreted substrates in a secretion-competent state. One such protein, CesT, serves a chaperone function for the enteropathogenic Escherichia coli (EPEC) translocated intimin receptor (Tir) protein, which confers upon EPEC the ability to alter host cell morphology following intimate bacterial attachment. Using a combination of complementary biochemical approaches, functional domains of CesT that mediate intermolecular interactions, involved in both chaperone-chaperone and chaperone-substrate associations, were determined. The CesT N-terminal is implicated in chaperone dimerization, whereas the amphipathic alpha-helical region of the C-terminal, is intimately involved in substrate binding. By functional complementation of chaperone domains using the Salmonella SicA chaperone to generate chaperone chimeras, we show that CesT-Tir interaction proceeds by a mechanism potentially common to other type III secretion system chaperones.

This chapter presents an overview on pathogenic organisms. Lan and Reeves describe the evolution of three important groups of enteric pathogens, Salmonella enterica, Escherichia coli (including Shigella), and Yersinia spp. At one time, the genus Salmonella was divided into more than 2,000 species based on surface and flagella antigens, but these are now considered to be one species, S. enterica, which is subdivided into seven subspecies and 2,501 serovars. Yersinia pestis is a recently emerged clone that shares 99.7% identity in 16S ribosomal RNA with Y. pseudotuberculosis and might more properly called Y. pseudotuberculosis Pestis. This clone has acquired additional virulence factors such as Yersinia murine toxin (Ymt) and plasminogen activator (Pla) that allow it to colonize the flea gut, be transmitted to a new host via biting, and disseminate hematogenously from the infected site. Clonal associations probably reflect the historical or prehistorical dissemination of M. tuberculosis with human migrations, similar to the associations that have been established for H. pylori. The great diversity of fungi, among which pathogenic species are dispersed across three phyla, plus the lack of information on virulence factors and the pathogenesis of fungal infections make the promulgation of generalized concepts for the evolution of fungal pathogens extremely difficult, if not impossible. In some cases, such as Legionnaires’ disease, archived clinical specimens may reveal that the disease existed decades ago but appropriate diagnostic techniques did not yet exist.

Virulence of Vibrio vulnificus correlates with changes in colony morphology that are indicative of a reversible phase variation for expression of capsular polysaccharide (CPS). Encapsulated variants are virulent with opaque colonies, whereas phase variants with reduced CPS expression are attenuated and are translucent. Using Tn phoA mutagenesis, we identified a V . vulnificus CPS locus, which included an upstream ops element, a wza gene ( wza Vv ), and several open reading frames with homology to CPS biosynthetic genes. This genetic organization is characteristic of group 1 CPS operons. The wza gene product is required for transport of CPS to the cell surface in Escherichia coli . Polar transposon mutations in wza Vv eliminated expression of downstream biosynthetic genes, confirming operon structure. On the other hand, nonpolar inactivation of wza Vv was specific for CPS transport, did not alter CPS biosynthesis, and could be complemented in trans . Southern analysis of CPS phase variants revealed deletions or rearrangements at this locus. A survey of environmental isolates indicated a correlation between deletions in wza Vv and loss of virulent phenotype, suggesting a genetic mechanism for CPS phase variation. Full virulence in mice required surface expression of CPS and supported the essential role of capsule in the pathogenesis of V . vulnificus .

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is responsible for outbreaks of bloody diarrhea and hemolytic-uremic syndrome in many countries. EHEC virulence mechanisms include the production of Shiga toxins (Stx) and formation of attaching and effacing (AE) lesions on intestinal epithelial cells. We recently reported that genes involved in the formation of the AE lesion were regulated by quorum sensing through autoinducer-2, which is synthesized by the product of the luxS gene. In this study we hybridized an E. coli gene array with cDNA synthesized from RNA that was extracted from EHEC strain 86-24 and its isogenic luxS mutant. We observed that 404 genes were regulated by luxS at least fivefold, which comprises approximately 10% of the array genes; 235 of these genes were up-regulated and 169 were down-regulated in the wild-type strain compared to in the luxS mutant. Down-regulated genes included several involved in cell division, as well as ribosomal and tRNA genes. Consistent with this pattern of gene expression, the luxS mutant grows faster than the wild-type strain (generation times of 37.5 and 60 min, respectively, in Dulbecco modified Eagle medium). Up-regulated genes included several involved in the expression and assembly of flagella, motility, and chemotaxis. Using operon:: lacZ fusions to class I, II, and III flagellar genes, we were able to confirm this transcriptional regulation. We also observed fewer flagella by Western blotting and electron microscopy and decreased motility halos in semisolid agar in the luxS mutant. The average swimming speeds for the wild-type strain and the luxS mutant are 12.5 and 6.6 μm/s, respectively. We also observed an increase in the production of Stx due to quorum sensing. Genes encoding Stx, which are transcribed along with λ-like phage genes, are induced by an SOS response, and genes involved in the SOS response were also regulated by quorum sensing. These results indicate that quorum sensing is a global regulatory mechanism for basic physiological functions of E. coli as well as for virulence factors.

Enteropathogenic Escherichia coli (EPEC) strains that carry the EPEC adherence factor (EAF) plasmid were screened for the presence of different EAF sequences, including those of the plasmid-encoded regulator ( per ). Considerable variation in gene content of EAF plasmids from different strains was seen. However, bfpA , the gene encoding the structural subunit for the bundle-forming pilus, bundlin, and per genes were found in 96.8% of strains. Sequence analysis of the per operon and its promoter region from 15 representative strains revealed that it is highly conserved. Most of the variation occurs in the 5′ two-thirds of the perA gene. In contrast, the C-terminal portion of the predicted PerA protein that contains the DNA-binding helix-turn-helix motif is 100% conserved in all strains that possess a full-length gene. In a minority of strains including the O119:H2 and canine isolates and in a subset of O128:H2 and O142:H6 strains, frameshift mutations in perA leading to premature truncation and consequent inactivation of the gene were identified. Cloned perA , - B , and - C genes from these strains, unlike those from strains with a functional operon, failed to activate the LEE1 operon and bfpA transcriptional fusions or to complement a per mutant in reference strain E2348/69. Furthermore, O119, O128, and canine strains that carry inactive per operons were deficient in virulence protein expression. The context in which the perABC operon occurs on the EAF plasmid varies. The sequence upstream of the per promoter region in EPEC reference strains E2348/69 and B171-8 was present in strains belonging to most serogroups. In a subset of O119:H2, O128:H2, and O142:H6 strains and in the canine isolate, this sequence was replaced by an IS 1294 -homologous sequence.

The pathogenicity island termed the locus of enterocyte effacement (LEE) is found in diverse attaching and effacing pathogens associated with diarrhea in humans and other animal species. To explore the relation of variation in LEE sequences to host specificity and genetic lineage, we determined the nucleotide sequence of the LEE region from a rabbit diarrheagenic Escherichia coli strain RDEC-1 (O15:H−) and compared it with those from human enteropathogenic E. coli (EPEC, O127:H6) and enterohemorrhagic E. coli (EHEC, O157:H7) strains. Differing from EPEC and EHEC LEEs, the RDEC-1 LEE is not inserted at selC and is flanked by an IS2 element and the lifA toxin gene. The RDEC-1 LEE contains a core region of 40 open reading frames, all of which are shared with the LEE of EPEC and EHEC. orf3 and the ERIC (enteric repetitive intergenic consensus) sequence present in the LEEs of EHEC and EPEC are absent from the RDEC-1 LEE. The predicted promoters of LEE1, LEE2, LEE3, tir , and LEE4 operons are highly conserved among the LEEs, although the upstream regions varied considerably for tir and the crucial LEE1 promoter, suggesting differences in regulation. Among the shared genes, high homology (>95% identity) between the RDEC-1 and the EPEC and EHEC LEEs at the predicted amino acid level was observed for the components of the type III secretion apparatus, the Ces chaperones, and the Ler regulator. In contrast, more divergence (66 to 88% identity) was observed in genes encoding proteins involved in host interaction, such as intimin (Eae) and the secreted proteins (Tir and Esps). A comparison of the highly variable genes from RDEC-1 with those from a number of attaching and effacing pathogens infecting different species and of different evolutionary lineages was performed. Although RDEC-1 diverges from some human-infecting EPEC and EHEC, most of the variation observed appeared to be due to evolutionary lineage rather than host specificity. Therefore, much of the observed hypervariability in genes involved in pathogenesis may not represent specific adaptation to different host species.

Regulation of virulence gene expression in enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) is incompletely understood. In EPEC, the plasmid-encoded regulator Per is required for maximal expression of proteins encoded on the locus of enterocyte effacement (LEE), and a LEE-encoded regulator (Ler) is part of the Per-mediated regulatory cascade upregulating the LEE2 , LEE3 , and LEE4 promoters. We now report that Ler is essential for the expression of multiple LEE-located genes in both EPEC and EHEC, including those encoding the type III secretion pathway, the secreted Esp proteins, Tir, and intimin. Ler is therefore central to the process of attaching and effacing (AE) lesion formation. Ler also regulates the expression of LEE-located genes not required for AE-lesion formation, including rorf2 , orf10 , rorf10 , orf19 , and espF , indicating that Ler regulates additional virulence properties. In addition, Ler regulates the expression of proteins encoded outside the LEE that are not essential for AE lesion formation, including TagA in EHEC and EspC in EPEC. Δ ler mutants of both EPEC and EHEC show altered adherence to epithelial cells and express novel fimbriae. Ler is therefore a global regulator of virulence gene expression in EPEC and EHEC.

In a study carried out in small-town and rural primary health care centers in southwestern Nigeria, 330 Escherichia coli strains isolated from 187 children with diarrhea and 144 apparently healthy controls were examined for virulence traits. Based on the results of colony blot hybridization, strains were categorized as enteropathogenic E. coli (1.8%), enterotoxigenic E. coli (2.4%), enteroinvasive E. coli (1.2%), enterohemorrhagic E. coli (0.6%), enteroaggregative E. coli (10.3%), diffusely adherent E. coli (7.9%), cell-detaching E. coli (6.9%), and cytolethal distending toxin-producing E. coli (0.9%). E. coli strains that hybridized with a Shiga toxin gene probe but lacked other characteristics usually present in enterohemorrhagic E. coli constituted 8.4% of the isolates. Ninety-seven E. coli isolates adhered to HEp-2 cells in an aggregative fashion but did not hybridize with any of the probes employed in the study. Overall the pathotypes, apart from cytolethal distending toxin-producing E. coli , were recovered both from children with diarrhea and from children without diarrhea, though to a lower extent from the healthy children. All diarrheagenic E. coli strains were associated with diarrhea ( P < 0.02). Heat-stable-enterotoxin-producing enterotoxigenic E. coli showed significant association with diarrhea ( P < 0.02), as did strains that demonstrated aggregative adherence to HEp-2 cells ( P < 0.04), but not those that hybridized with the CVD432 enteroaggregative probe.

The locus of enterocyte effacement (LEE) pathogenicity island of enterohemorrhagic Escherichia coli (EHEC) O157:H7 possesses the same genes in identical order and orientation as the LEE of enteropathogenic E. coli (EPEC) O127:H6 but is unable to form attaching and effacing (A/E) lesions or to secrete Esp proteins when it is cloned in an E. coli K-12 background. The A/E phenotype could not be restored by trans complementation with a variety of cloned EPEC LEE fragments, suggesting functional and/or regulatory differences between the LEE pathogenicity islands of EPEC O127:H6 and EHEC O157:H7.

Enteropathogenic Escherichia coli (EPEC) is the prototype organism of a group of pathogenic Gram-negative bacteria that cause attaching and effacing (AE) intestinal lesions. All EPEC genes necessary for the AE phenotype are encoded within a 35.6 kb pathogenicity island termed the locus of enterocyte effacement (LEE). The LEE encodes 41 predicted open reading frames (ORFs), including components of a type III secretion apparatus and secreted molecules involved in the disruption of the host cell cytoskeleton. To initiate our studies on regulation of genes within the LEE, we determined the genetic organization of the LEE, defining transcriptional units and mapping transcriptional start points. We found that components of the type III secretion system are transcribed from three polycistronic operons designated LEE1, LEE2 and LEE3. The secreted Esp molecules are part of a fourth polycistronic operon designated LEE4. Using reporter gene fusion assays, we found that the previously described plasmid-encoded regulator (Per) activated operons LEE1, LEE2 and LEE3, and modestly increased the expression of LEE4 in EPEC. Using single-copy lacZ fusions in K-12-derived strains, we determined that Per only directly activated the LEE1:lacZ fusion, and did not directly activate the other operons. Orf1 of the LEE1 operon activated the expression of single-copy LEE2:lacZ and LEE3:lacZ fusions in trans and modestly increased the expression of LEE4:lacZ in K-12 strains. Orf1 was therefore designated Ler, for LEE-encoded regulator. Thus, the four polycistronic operons of the LEE that encode type III secretion components and secreted molecules are now included in the Per regulon, where Ler participates in this novel regulatory cascade in EPEC.

The pathophysiology of enteropathogenic Escherichia coli (EPEC) diarrhea remains uncertain. In vitro, EPEC stimulates a rapid increase in short-circuit current ( I sc ) across Caco-2 cell monolayers coincident with intimate attaching and effacing (A/E) bacterial adhesion. This study has examined the roles of specific EPEC virulence proteins in this I sc response. EPEC genes encoding EspA, EspB, and EspD, essential for signal transduction in host cells and A/E activity, were also required for modulation of Caco-2 electrolyte transport.

Enteropathogenic Escherichia coli (EPEC) encode a type III secretion system located on a pathogenicity island known as the locus for enterocyte effacement. Four proteins are known to be exported by this type III secretion system – EspA, EspB and EspD required for subversion of host cell signal transduction pathways and a translocated intimin receptor protein (Tir) required for intimin-mediated intimate attachment and attaching and effacing lesion formation. The espA gene is located within the locus for enterocyte effacement and the EspA polypeptide from the prototype EPEC strain E2348/69 (O127:H6) has recently been shown to be a component of a filamentous structure involved in bacteria-host cell interaction and locus for enterocyte effacement-encoded protein translocation involved in attaching and effacing lesion formation. In this study we have extended our investigation of EspA to strains belonging to other classical EPEC serotypes. DNA sequencing demonstrated that the espA gene from the different EPEC strains share at least 65% DNA identity. In addition, we detected morphologically and antigenically similar EspA filaments in all but one of the bacterial strains examined including recombinant, non-pathogenic E. coli expressing espA from a cloned locus for enterocyte effacement region (HB101(pCVD462)).

Enteropathogenic (EPEC) and enterohaemorrhagic Escherichia coli (EHEC) constitute a significant risk to human health worldwide. Both pathogens colonize the intestinal mucosa and, by subverting intestinal epithelial cell function, produce a characteristic histopathological feature known as the 'attaching and effacing' (A/E) lesion. Although EPEC was the first E. coli to be associated with human disease in the 1940s and 1950s, it was not until the late 1980s and early 1990s that the mechanisms and bacterial gene products used to induce this complex brush border membrane lesion and diarrhoeal disease started to be unravelled. During the past few months, there has been a burst of new data that have revolutionized some basic concepts of the molecular basis of bacterial pathogenesis in general and EPEC pathogenesis in particular. Major breakthroughs and developments in the genetic basis of A/E lesion formation, signal transduction, protein translocation, host cell receptors and intestinal colonization are highlighted in this review.

All proteins involved in the attachment and effacement lesion produced by enteropathogenic Escherichia coli (EPEC) and Shiga-toxin producing E. coli (STEC) are encoded by the locus of enterocyte effacement (LEE). We studied the presence and insertion site of the LEE in different EPEC and STEC strains. In serotypes O119:H6/H-, O55:H6, O55:H7, O142:H6, O111ac:H9/H-, O111ab:H9/H- LEE is inserted downstream of selC as previously described for EPEC O127:H6 and STEC O157:H7. In serotypes O111ac:H8/H- and O26:H11/H- the LEE is inserted in pheU as previously described for STEC O26:H-. However in EPEC from serotype O111ab:H25 the LEE is not inserted in either site suggesting a third insertion site in the K12 chromosome. We also cloned fragments of 2.3 kb and 1.0 kb from the right and left hand sides of the LEE of a O111ac:H- strain and identified additional insertion sequences on these LEE fragments, suggesting that the LEE may be larger and may have undergone more recombination events in these serotypes.

I thank Stuart Knutton, Gad Frankel, Ilan Rosenshine, Trinad Chakraborty and Michael Donnenberg for sharing unpublished or `in press' data, and Simon Elliott for assistance with Fig. 1Fig. 1. Research in my laboratory is supported by NIH research grants AI 21657 and AI 41325.

Attaching and effacing (A/E) lesion formation is central to enteropathogenic Escherichia coli (EPEC) pathogenesis. In vitro experiments with human epithelial cell lines have implicated virulence plasmid-encoded bundle-forming pili (BFP) in initial binding and intimin in intimate attachment and A/E lesion formation. This study investigated the role of BFP and intimin in EPEC interactions with pediatric small intestinal biopsy tissue in in vitro organ culture. Organ culture infections (2 to 8 h) were performed with E2348/69 (a wild-type EPEC O127:H6 clinical isolate) and E2348/69 derivatives including CVD206 ( eae deficient), CVD206(pCVD438) ( eae -complemented CVD206), CVD206(pCVD438/01) (expressing intimin, which is nonfunctional due to a single amino acid substitution), JPN15 (spontaneous EPEC adherence factor virulence plasmid-cured E2348/69), and 31-6-1(1) (E2348/69 with a Tn phoA insertion inactivation mutation in the virulence plasmid-encoded bfpA gene). Scanning and transmission electron microscopy revealed that after 8 h E2348/69 and CVD206(pCVD438) (both Int + BFP + ) adhered to all specimens, causing A/E lesions with surrounding microvillous elongation. JPN15 and 31-6-1(1) (both Int + BFP − ) adhered and caused A/E lesions although bacteria adhered in “flat,” two-dimensional groups. CVD206 and CVD206(pCVD438/01) (both Int − BFP + ) did not adhere to any sample, and no pathological tissue changes were seen. Thus, in human intestinal organ culture, BFP do not appear to be involved in the initial stages of EPEC nonintimate adhesion but are implicated in the formation of complex, three-dimensional colonies via bacterium-bacterium interactions. Intimin appears to play an essential role in establishing colonization of EPEC on pediatric small intestinal tissue.

Enteropathogenic Escherichia coli uses a type III secretion apparatus to deliver proteins essential for pathogenesis to the host epithelium. Several proteins have been detected in culture supernatants of the prototype EPEC strain E2348/69 and three of these, EspA, EspB, and EspD, use type III machinery for export. Here, we report the identification and characterization of CesD, a protein required for proper EspB and EspD secretion. CesD shows sequence homology to chaperone proteins from other type III secretion pathways. Based on this, we hypothesize that CesD may function as a secretion chaperone in EPEC. A mutation in cesD abolished EspD secretion into culture supernatants and reduced the amount of secreted EspB, but had little effect on the amount of secreted EspA. The mutant strain was negative for both FAS and Tir phosphorylation, consistent with the previously described roles for EspB and EspD in EPEC pathogenesis. CesD was shown to interact with EspD but not EspB or EspA. CesD was detected in the bacterial cytosol, and, surprisingly, a substantial amount of the protein was also found to be associated with the inner membrane. Thus, although CesD has some attributes that are similar to other type III secretion chaperones, its membrane localization separates it from previously described members of this family.

Enterohemorrhagic Escherichia coli has been responsible for an increasing number of large food-borne outbreaks of bloody diarrhea and hemolytic uremic syndrome. Recent developments in our understanding of the pathogenesis of disease due to enterhemorrhagic E. coli include the description of a pathogenicity island, a type III secretion system and potential plasmid-encoded virulence factors. Recent developments in our understanding of the epidemiology include a recognition of a widening spectrum of vehicles.

An outbreak of gastrointestinal illness with clinical and epidemiologic features of enterotoxigenic Escherichia coli (ETEC) occurred among patrons of a restaurant during April 1991. Illnesses among several groups of patrons were characterized by diarrhea (100%) and cramps (79%-88%) lasting a median of 3-5 days. Median incubation periods ranged from 50 to 56 h. A nonmotile strain of E. coli (E. coli O39), which was negative for heat-labile (LT) and heat-stable (STa, STb) ETEC toxins, was isolated only from ill patrons. This organism produced enteroaggregative E. coli heat-stable enterotoxin 1 and contained the enteropathogenic E. coli gene locus for enterocyte effacement; it did not display mannose-resistant adherence, but produced attaching and effacing lesions in the absence of mannose on cultured HEp-2 cells. E. coli that are not part of highly characterized but narrowly defined groups may be important causes of foodborne illness.

Longus is a long pilus produced by human enterotoxigenic Escherichia coli (ETEC) which shares significant structural and biochemical features with class-B type-4 pili. These pili include the toxin-coregulated pilus (TCP) of Vibrio cholerae, the bundle-forming pilus (BFP) of enteropathogenic E. coli and both longus and the colonization factor antigen III (CFA/III) of ETEC. These pili are produced under defined growth conditions indicating that they are under the control of different regulatory elements. While TCP is chromosomally encoded, the remaining pili are encoded on large virulence plasmids. Longus and CFA/III are closely related pili although certain DNA and protein differences also exist between them. This may account for the differences in the regulation, surface presentation, antigenicity, and prevalence of these two pilins among ETEC. Neighboring lngA, a second open reading frame termed lngB was found which encodes a protein with significant homology to proteins which are part of a type-II secretory system such as XcpV, OutC, and PulO of Pseudomonas aeruginosa, Erwinia chrysanthemi, and Klebsiella pneumoniae, respectively. This suggests that lngB may be an accessory gene involved in biogenesis of longus.

Gnotobiotic piglets inoculated with Escherichia coli O157:H7, its luxS mutant derivative, or nonpathogenic E. coli were evaluated for attaching and effacing lesions. Although no differences in clinical symptoms were seen between pigs inoculated with the parent and those inoculated with the luxS mutant, the luxS mutant-inoculated pigs had a lower frequency of attaching and effacing lesions in the spiral colon than parent strain-inoculated pigs.

Life-threatening intestinal and systemic effects of the Shiga toxins produced by enterohemorrhagic Escherichia coli (EHEC) require toxin uptake and transcytosis across intestinal epithelial cells. We have recently demonstrated that EHEC infection of intestinal epithelial cells stimulates toxin macropinocytosis, an actin-dependent endocytic pathway. Host actin rearrangement necessary for EHEC attachment to enterocytes is mediated by the type 3 secretion system which functions as a molecular syringe to translocate bacterial effector proteins directly into host cells. Actin-dependent EHEC attachment also requires the outer membrane protein intimin, a major EHEC adhesin. Here, we investigate the role of type 3 secretion in actin turnover occurring during toxin macropinocytosis. Toxin macropinocytosis is independent of EHEC type 3 secretion and intimin attachment. EHEC soluble factors are sufficient to stimulate macropinocytosis and deliver toxin into enterocytes in vitro and in vivo; intact bacteria are not required. Intimin-negative enteroaggregative Escherichia coli (EAEC) O104:H4 robustly stimulate Shiga toxin macropinocytosis into intestinal epithelial cells. The apical macropinosomes formed in intestinal epithelial cells move through the cells and release their cargo at these cells’ basolateral sides. Further analysis of EHEC secreted proteins shows that a serine protease EspP alone is able to stimulate host actin remodeling and toxin macropinocytosis. The observation that soluble factors, possibly serine proteases including EspP, from each of two genetically distinct toxin-producing strains, can stimulate Shiga toxin macropinocytosis and transcellular transcytosis alters current ideas concerning mechanisms whereby Shiga toxin interacts with human enterocytes. Mechanisms important for this macropinocytic pathway could suggest new potential therapeutic targets for Shiga toxin-induced disease.

An unusual mutant of enteropathogenic E. coli (EPEC), deficient in its ability to invade host cells, was evaluated. The gene interrupted by the transposon in this mutant was located within a region of the EPEC chromosome devoted to secretion of proteins required for signal transduction. The mutant did not secrete detectable levels of the EspB protein, previously shown to be required for attaching and effacing, and did not induce detectable tyrosine phosphorylation of a 90 kDa host cell protein, previously associated with attaching and effacing and invasion. No quantitative or qualitative defect in the ability of the mutant to induce attaching and effacing effects was observed. Moreover, attaching and effacing by wild-type EPEC was unaffected by high doses of the tyrosine kinase inhibitor genistein. These results indicate that attaching and effacing activity can occur in the absence of detectable EspB secretion and tyrosine kinase mediated signal transduction.