Functional studies of intimin in vivo and ex vivo: implications for host specificity and tissue tropism.

Intimin is an outer-membrane adhesin that is essential for colonization of the host gastrointestinal tract by attaching and effacing pathogens including enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR). The N-terminus of intimin from the different strains is highly conserved while the C-terminus, which harnesses the active receptor-binding site, shows sequence and antigenic polymorphism. This diversity was used to define a number of distinct intimin types, the most common of which are alpha, beta and gamma. Intimin binds the type III secretion system effector protein Tir. However, a large body of evidence suggests that intimin also binds a host-cell-encoded receptor(s) (Hir), and interaction of different intimin types with Hir contributes to tissue and host specificity. The aims of this study were to compare the activity of the major intimin types (alpha, beta and gamma) in vivo and ex vivo, using the CR mouse model and in vitro organ culture (IVOC), and to determine their exchangeability. The results confirm that intimin gamma is not functional in the CR mouse model. In the pig, intimin beta can substitute for EPEC intimin alpha but when placed in an EHEC O157 : H7 background it does not produce an intimin alpha-like tropism, although some adhesion to the small and large intestine was observed. In contrast, in human IVOC, intimin beta in an EHEC background produces small intestinal colonization in a similar manner to intimin alpha.

Comparison of colonization dynamics and pathology of mice infected with enteropathogenic Escherichia coli, enterohaemorrhagic E. coli and Citrobacter rodentium.

Enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic E. coli (EHEC) and Citrobacter rodentium (CR) colonize the gastrointestinal tract epithelium via attaching and effacing lesions. While humans are believed to be the only living reservoir of typical EPEC and EHEC to have border host specificity, CR is a restricted mouse pathogen. Recently, conflicting conclusions were reported concerning the utility of a murine model to study mechanisms of EPEC and EHEC colonization and infection. We therefore aimed to compare colonization dynamics of EPEC, EHEC and CR, together with a commensal E. coli (Nissle) as a control, in the murine. We show that all strains are equally shed in stools over the first 48 h post inoculation. However, while the CR population then rapidly expanded the EPEC, EHEC and Nissle populations quickly declined to a level just above detection. We conclude that following oral inoculation EPEC and EHEC develop a commensal, rather than pathogenic, interaction within the mouse host.

Identification and characterization of EspK, a type III secreted effector protein of enterohaemorrhagic Escherichia coli O157:H7.

Enterohaemorrhagic Escherichia coli (EHEC) causes bloody diarrhoea in humans and deploys a type III secretion system (T3SS) encoded by the locus of enterocyte effacement to elicit the formation of attaching and effacing (AE) lesions on intestinal epithelia. Here, we report the identification of a new secreted substrate of this system, z1829, which is encoded by cryptic prophage CP-933N. Elevated secretion of a beta-lactamase-z1829 fusion protein was detected upon mutation of sepD in EHEC O157:H7 and the fusion protein was translocated into infected epithelial cells in a T3SS-dependent manner; accordingly, we named the protein EspK. In common with the related Salmonella enterica type III secreted effector GogB, we observed that EspK localized to the cytoplasm when transiently expressed in COS-7 cells using EspK-specific antiserum. Inactivation of espK did not impair adherence or actin nucleation during infection of HeLa cells but affected persistence of EHEC O157:H7 in the intestines of orally inoculated calves. Inactivation of an orthologue of espK in the murine AE pathogen Citrobacter rodentium did not impair intestinal colonization in mice.

The enteropathogenic Escherichia coli type III secretion system effector Map binds EBP50/NHERF1: implication for cell signalling and diarrhoea.

Enteropathogenic Escherichia coli (EPEC) is the single most important contributor to child diarrhoea in developing countries. Nevertheless, the mechanism responsible for EPEC diarrhoea remains elusive. Using the yeast two-hybrid system to determine the target host cell protein of the EPEC type III secretion system effector Map led to identification of ezrin/radixin/moesin (ERM)-binding phosphoprotein 50 (EBP50), also known as Na+/H+ exchanger regulatory factor 1 (NHERF1). Protein interaction is mediated by the carboxy-terminal Thr-Arg-Leu (TRL) motif of Map and the PSD-95/Disk-large/ZO-1 domain 1 (PDZ1) of EBP50/NHERF1. Although EBP50/NHERF1 is recruited to site of EPEC adhesion in a Map-independent mechanism, co-immunoprecipitation and immunostaining revealed that Map binds to, induces proteolysis of, and colocalizes with EBP50/NHERF1 during infection of cultured epithelial cells. The TRL motif of Map was involved in Map-induced filopodia formation and brush border elongation on infected HeLa and Caco-2 cells respectively. As EBP50/NHERF1 regulates ion channels in the intestine we assessed the involvement of Map in diarrhoea using the Citrobacter rodentium mouse model of EPEC. We report significantly greater diarrhoea following infections with wild-type C. rodentium compared with C. rodentiumDeltamap. These results provide new insights into the mechanisms of EPEC diarrhoea.

Essential role of the type III secretion system effector NleB in colonization of mice by Citrobacter rodentium.

Attaching and effacing (A/E) pathogens are a significant cause of gastrointestinal illness in humans and animals. All A/E pathogens carry a large pathogenicity island, termed the locus for enterocyte effacement (LEE), which encodes a type III secretion system that translocates several effector proteins into host cells. To identify novel virulence determinants in A/E pathogens, we performed a signature-tagged mutagenesis screen in C57BL/6 mice by using the mouse A/E pathogen Citrobacter rodentium. Five hundred seventy-six derivatives of C. rodentium were tested in pools of 12 mutants. One attenuated mutant carried a transposon insertion in nleB, which encodes a putative effector of the LEE-encoded type III secretion system (T3SS). nleB is present in a genomic pathogenicity island that also encodes another putative effector, NleE, immediately downstream. Using translational fusions with beta-lactamase (TEM-1), we showed that both NleB and NleE were translocated into host cells by the LEE-encoded T3SS of enteropathogenic Escherichia coli. In addition, deletion of the gene encoding NleB in C. rodentium resulted in reduced colonization of mice in single infections and reduced colonic hyperplasia. In contrast, the deletion of other non-LEE-encoded effector genes in C. rodentium, nleC, nleD, or nleE, had no effect on host colonization or disease. These results suggest that nleB encodes an important virulence determinant of A/E pathogens.

The lpf gene cluster for long polar fimbriae is not involved in adherence of enteropathogenic Escherichia coli or virulence of Citrobacter rodentium.

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 EPECDeltalpfABCD(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 DeltalpfA(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.

Citrobacter rodentium of mice and man.

The major classes of enteric bacteria harbour a conserved core genomic structure, common to both commensal and pathogenic strains, that is most likely optimized to a life style involving colonization of the host intestine and transmission via the environment. In pathogenic bacteria this core genome framework is decorated with novel genetic islands that are often associated with adaptive phenotypes such as virulence. This classical genome organization is well illustrated by a group of extracellular enteric pathogens, which includes enteropathogenic Escherichia coli (EPEC), enterohaemorrhagic E. coli (EHEC) and Citrobacter rodentium, all of which use attaching and effacing (A/E) lesion formation as a major mechanism of tissue targeting and infection. Both EHEC and EPEC are poorly pathogenic in mice but infect humans and domestic animals. In contrast, C. rodentium is a natural mouse pathogen that is related to E. coli, hence providing an excellent in vivo model for A/E lesion forming pathogens. C. rodentium also provides a model of infections that are mainly restricted to the lumen of the intestine. The mechanism's by which the immune system deals with such infections has become a topic of great interest in recent years. Here we review the literature of C. rodentium from its emergence in the mid-1960s to the most contemporary reports of colonization, pathogenesis, transmission and immunity.

Distribution of espI among clinical enterohaemorrhagic and enteropathogenic Escherichia coli isolates.

Enterohaemorrhagic (EHEC) and enteropathogenic (EPEC) Escherichia coli are important diarrhoeagenic pathogens; infection is dependent on translocation of a number of type III effector proteins. Until recently all the known effectors were encoded on the LEE pathogenicity island, which also encodes the adhesin intimin and the type III secretion apparatus. Recently, a novel non-LEE effector protein, EspI/NleA, which is required for full virulence in vivo and is encoded on a prophage, was identified. The aim of this study was to determine the distribution of espI among clinical EHEC and EPEC isolates. espI was detected in 86 % and 53 % of LEE+ EHEC and EPEC strains, respectively. Moreover, the espI gene was more commonly found in patients suffering from a more severe disease.

Identification of a novel Citrobacter rodentium type III secreted protein, EspI, and roles of this and other secreted proteins in infection.

Citrobacter rodentium is a member of a group of pathogens that colonize the lumen of the host gastrointestinal tract via attaching and effacing (A/E) lesion formation. C. rodentium, which causes transmissible colonic hyperplasia in mice, is used as an in vivo model system for the clinically significant A/E pathogens enterohemorrhagic and enteropathogenic Escherichia coli. These bacteria all contain a pathogenicity island called the locus of enterocyte effacement (LEE), which encodes a type III secretion system that is designed to deliver effector proteins into eukaryotic host cells. These effectors are involved in the subversion of host eukaryotic cell functions to the benefit of the bacterium. In this study we used mutant strains to determine the effects of the C. rodentium LEE-encoded effectors EspF, EspG, EspH, and Map on virulence in the mouse model. In addition, we identified a novel secreted protein, EspI encoded outside the LEE, whose secretion is also dependent on a functional type III secretion system. Mutant strains with each of the effectors investigated were found to be outcompeted by wild-type bacteria in mixed-infection experiments in vivo, although the effects of EspF and EspH were only subtle. In single-infection experiments, we found that EspF, EspG, and EspH are not required for efficient colonization of the mouse colon or for the production of hyperplasia. In contrast, strains producing EspI and Map had significant colonization defects and resulted in dramatically reduced levels of hyperplasia, and they exhibited very different growth dynamics in mice than the wild-type strain exhibited.

SseA is a chaperone for the SseB and SseD translocon components of the Salmonella pathogenicity-island-2-encoded type III secretion system.

The type III secretion system (TTSS) encoded by the Salmonella pathogenicity island 2 (SPI-2) is required for bacterial replication inside macrophages and for systemic infection in mice. Many TTSS secreted proteins, including effectors and components of the translocon, require chaperones which promote their stability, prevent their premature interactions or facilitate their secretion. In this study, the function of the first gene (sseA) of one of the SPI-2 operons (sseA-G) was investigated. This operon includes genes that encode translocon components (SseB, SseC and SseD), translocated proteins (SseF and SseG) and putative chaperones (SscA and SscB). sseA encodes a 12.5 kDa protein with a C-terminal region with the potential to form a coiled-coil structure, but no sequence similarity to other proteins. Mutation of sseA results in severe virulence attenuation and an intracellular replication defect. It is shown here that SseA is not a secreted protein, but is required for SPI-2-dependent translocation of two effector proteins (SifA and PipB). Furthermore, the translocon components SseB and SseD were not detected in an sseA mutant strain. By using a yeast two-hybrid assay and column binding experiments, it is demonstrated that SseA interacts directly with SseB and SseD. These results indicate that SseA is a chaperone for SseB and SseD. The inability of an sseA mutant to assemble the SPI-2 TTSS translocon accounts for its high level of virulence attenuation in vivo. To the authors' knowledge, this is the first chaperone described for the SPI-2 TTSS.

Identification of a novel type IV pilus gene cluster required for gastrointestinal colonization of Citrobacter rodentium.

Citrobacter rodentium is used as an in vivo model system for clinically significant enteric pathogens such as enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). These pathogens all colonize the lumen side of the host gastrointestinal tract via attaching and effacing (A/E) lesion formation. In order to identify genes required for the colonization of A/E-forming pathogens, a library of signature-tagged transposon mutants of C. rodentium was constructed and screened in mice. Of the 576 mutants tested, 14 were attenuated in their ability to colonize the descending colon. Of these, eight mapped to the locus of enterocyte effacement (LEE), which is required for the formation of A/E lesions, underlying the importance of this mechanism for pathogenesis. Another mutant, P5H2, was found to have a transposon insertion in an open reading frame that has strong similarity to type IV pilus nucleotide-binding proteins. The region flanking the transposon insertion was sequenced, identifying a cluster of 12 genes that encode the first described pilus of C. rodentium (named colonization factor Citrobacter, CFC). The proteins encoded by cfc genes have identity to proteins of the type IV COF pilus of enterotoxigenic E. coli (ETEC), the toxin co-regulated pilus of Vibrio cholerae and the bundle-forming pilus of EPEC. A non-polar mutation in cfcI, complementation of this strain with wild-type cfcI and complementation of strain P5H2 with wild-type cfcH confirmed that these genes are required for colonization of the gastrointestinal tract by C. rodentium. Thus, CFC provides a convenient model to study type IV pilus-mediated pathogen-host interactions under physiological conditions in the natural colonic environment.

CesD2 of enteropathogenic Escherichia coli is a second chaperone for the type III secretion translocator protein EspD.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli are extracellular pathogens that employ a type III secretion system to export translocator and effector proteins, proteins which facilitates colonization of the mucosal surface of the intestine via formation of attaching and effacing (A/E) lesions. The genes encoding the proteins for A/E lesion formation are located on a pathogenicity island, termed the locus of enterocyte effacement (LEE), which contains eae encoding intimin as well as the type III secretion system and effector genes. Many type III secreted proteins are stabilized and maintained in a secretion-competent conformation in the bacterial cytosol by specific chaperone proteins. Three type III chaperones have been described thus far within the EPEC LEE region: CesD, for the translocator proteins EspB and EspD; CesT, for the effector proteins Tir and Map; and CesF, for EspF. In this study we report the characterization of CesD2 (previously Orf27), a second LEE-encoded chaperone for EspD. We show specific CesD2-EspD protein interaction which appears to be necessary for proper EspD secretion in vitro and pathogenesis in vivo as demonstrated in the A/E-lesion-forming mouse pathogen Citrobacter rodentium.

Tyrosine residues at the immunoglobulin-C-type lectin inter-domain boundary of intimin are not involved in Tir-binding but implicated in colonisation of the host.

Intimin is an outer membrane adhesion molecule involved in bacterial adhesion to intestinal epithelium by several human and animal enteric pathogens, including enteropathogenic and enterohaemorrhagic Escherichia coli and Citrobacter rodentium. Intimin binds to the translocated intimin receptor, Tir, which is delivered to the plasma membrane of the host cell by a type III protein translocation system. Intimin is also implicated in binding to a host cell-encoded intimin receptor (Hir). The receptor-binding activity of intimin resides within the carboxy terminus 280 amino acids (Int280) of the polypeptide. Structural analysis of this region revealed two immunoglobulin-like domains, the second of which forms a number of contacts with the distal C-type lectin-like module. Specific orientation differences at this inter-domain boundary, which consists of several tyrosine residues, were detected between the crystal and solution structures. In this study, we determined the influence of site-directed mutagenesis of each of four tyrosine residues on intimin-Tir interactions and on intimin-mediated intimate attachment. The mutant intimins were also studied using a variety of in vitro and in vivo infection models. The results show that three of the four Tyr, although not essential for A/E lesion formation in vitro, are required for efficient colonisation of the mouse host following oral challenge.