Emergence of a 'hyperinfectious' bacterial state after passage of Citrobacter rodentium through the host gastrointestinal tract

Citrobacter rodentium belongs to a family of human and animal enteric pathogens that includes the clinically significant enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). These pathogens exploit attaching and effacing (A/E) lesions to colonize the host gastrointestinal tract. However, both EHEC and EPEC are poorly pathogenic in mice. In contrast, C. rodentium, which is genetically highly related to E. coli, relies on A/E lesion formation as an essential step in both colonization and infection of the murine mucosa, providing an excellent in vivo model. In this study we have used bioluminescence imaging (BLI) to investigate the organ specificity and dynamics of colonization of mice by LB-grown and mouse-passaged C. rodentium in situ and in real time. We have demonstrated the appearance of a 'hyperinfectious' state after passage of C. rodentium through the murine gastrointestinal tract. The 'hyperinfectious' state was found to dramatically reduce the dose required to infect secondary individuals, and also influenced the tissue distribution of colonizing bacteria, removing the requirement for primary colonization of the caecal patch. In addition, the 'hyperinfectious' phenotype was found to be transient with one overnight passage in rich medium sufficient to return C. rodentium to 'culture' infectivity.     

Tir phosphorylation and Nck/N-WASP recruitment by enteropathogenic and enterohaemorrhagic Escherichia coli during ex vivo colonization of human intestinal mucosa is different to cell culture models

Tir, the translocated intimin receptor of enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) and Citrobacter rodentium, is translocated into the host cell by a filamentous type III secretion system. Epithelial cell culture has demonstrated that Tir tyrosine phosphorylation is necessary for attaching effacing (A/E) lesion formation by EPEC and C. rodentium, but is not required by EHEC O157:H7. Recent in vivo work on C. rodentium has reported that Tir translocation, but not its phosphorylation, is necessary for colonization of the mouse colon. In this study we investigated the involvement of Tir and its tyrosine phosphorylation in EPEC and EHEC human intestinal colonization, N-WASP accumulation and F-actin recruitment using in vitro organ culture (IVOC). We showed that both EPEC and EHEC Tir are translocated into human intestinal epithelium during IVOC and that Tir is necessary for ex vivo intestinal colonization by both EPEC and EHEC. EPEC, but not EHEC, Tir is tyrosine phosphorylated but Tir phosphorylation-deficient mutants still colonize intestinal explants. While EPEC Tir recruits the host adaptor protein Nck to initiate N-WASP-Arp2/3-mediated actin polymerization, Tir derivatives deficient in tyrosine phosphorylation recruit N-WASP independently of Nck indicating the presence of a tyrosine phosphorylation-independent mechanism of A/E lesion formation and actin recruitment ex vivo by EPEC in man.     

Citrobacter rodentium translocated intimin receptor (Tir) is an essential virulence factor needed for actin condensation,intestinal colonization and colonic hyperplasia in mice

Citrobacter rodentium infection of mice serves as a relevant small animal model to study enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC) infections in man. Enteropathogenic E. coli and EHEC translocate Tir into the host cytoplasmic membrane, where it serves as the receptor for the bacterial adhesin intimin and plays a central role in actin condensation beneath the adherent bacterium. In this report, we examined the function of C. rodentium Tir both in vitro and in vivo. Similar to EPEC, C. rodentium Tir is tyrosine phosphorylated and is essential for actin condensation. Citrobacter Tir and EPEC Tir are functionally interchangeable and both require tyrosine phosphorylation to mediate actin rearrangements. In contrast, Citrobacter Tir supports actin nucleation in EHEC independent of tyrosine phosphorylation, while EHEC Tir cannot replace Citrobacter Tir for this function. This indicates that C. rodentium and EPEC use an actin nucleating mechanism different from EHEC. We also found that Tir is expressed and translocated into mouse enterocytes in vivo by C. rodentium during infections. This represents the first direct demonstration of a type III effector translocated in vivo into a natural host by any pathogen. In addition, we showed that Tir, but not its tyrosine phosphorylation, is essential for C. rodentium to colonize the large bowel and induce attaching/effacing (A/E) lesions and colonic hyperplasia in mice, and that both EPEC Tir and EHEC Tir can substitute for Citrobacter Tir for these activities in vivo. These results thus demonstrate that Tir is an essential virulence factor in this infection model. The data also show that the function of Tir tyrosine phosphorylation and its subsequent actin nucleating activity are not essential for C. rodentium colonization of the mouse gut nor for inducing A/E lesions and colonic hyperplasia, thereby uncoupling colonization and disease from actin condensation for this A/E pathogen.     

Generation of Escherichia coli intimin derivatives with differing biological activities using site-directed mutagenesis of the intimin C-terminus domain

Intimins, encoded by eae genes, are outer membrane proteins involved in attaching–effacing (A/E) lesion formation and host cell invasion by pathogenic bacteria, including enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium . A series of intimins, harbouring specific mutations close to the C-terminus, were constructed using pCVD438, which encodes the eae gene from EPEC strain E2348/69. These mutant plasmids were introduced into EPEC strain CVD206 and C. rodentium strain DBS255, which both contain deletion mutations in their eae genes. CVD206, CVD206(pCVD438) and CVD206(pCVD438) derivatives were assessed for their ability to promote A/E lesion formation or invasion of HEp-2 cells and to induce A/E lesions on fresh human intestinal in vitro organ cultures (IVOC). The pathogenicity of C. rodentium DBS255 harbouring these plasmid derivatives was also studied in mice. Here, we report that intimin-mediated A/E lesion formation can be segregated from intimin-mediated HEp-2 cell invasion. Moreover, adherence to IVOC, EPEC-induced microvillus elongation and colonization of the murine intestine by C. rodentium were also modulated by the modified intimins.     

Organ specificity, colonization and clearance dynamics in vivo following oral challenges with the murine pathogen Citrobacter rodentium

Citrobacter rodentium belongs to a family of human and animal enteric pathogens that includes the clinically significant enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). These pathogens use attaching and effacing (A/E) lesions to colonize the host gastrointestinal tract. In this study we have used bioluminescence imaging (BLI) to investigate the organ specificity, dynamics of colonization and clearance of mice by C. rodentium in situ and in real time. The bioluminescent C. rodentium derivative, strain ICC180, expresses the luxCDABE operon from the entemopathogenic nematode symbiont Photorhabdus luminescens and light levels accurately reflect bacterial numbers both in vitro and in vivo. We have demonstrated that primary colonization of the mouse by C. rodentium takes place within the caecum, specifically within the specialized patch of lymphoid tissue known as the caecal patch. Following colonization of the caecum C. rodentium established a colonic infection. Clearance of C. rodentium ICC180 parallels the colonization dynamics, i.e. the caecum was first to be cleared followed by the colon. A bioluminescent eae (encoding the outer membrane adhesin intimin) C. rodentium mutant failed to establish long-term colonization, although low levels of bacteria could be recovered for up to 3 days post challenge from the caecum.     

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.     

Sec24 interaction is essential for localization and virulence-associated function of the bacterial effector protein NleA

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) are food-borne pathogens that cause severe diarrhoeal disease in humans. Citrobacter rodentium is a related mouse pathogen that serves as a small animal model for EPEC and EHEC infections. EPEC, EHEC and C. rodentium translocate bacterial virulence proteins directly into host cells via a type III secretion system (T3SS). Non-LEE-encoded effector A (NleA) is a T3SS effector that is common to EPEC, EHEC and C. rodentium and is required for bacterial virulence. NleA localizes to the host cell secretory pathway and inhibits vesicle trafficking by interacting with the Sec24 subunit of mammalian coatamer protein II complex (COPII). Mammalian cells express four paralogues of Sec24 (Sec24A-D), which mediate selection of cargo proteins for transport and possess distinct, but overlapping cargo specificities. Here, we show that NleA binds Sec24A-D with two distinct mechanisms. An NleA protein variant with greatly diminished interaction with all Sec24 paralogues does not properly localize, does not inhibit COPII-mediated vesicle budding, and does not confer virulence in the mouse infection model. Together, this work provides strong evidence that the interaction and inhibition of COPII by NleA is an important aspect of EPEC- and EHEC-mediated disease.     

Attaching effacing Escherichia coli and paradigms of Tir-triggered actin polymerization: getting off the pedestal

Enteropathogenic and enterohaemorrhagic Escherichia coli (EPEC and EHEC) colonize the gut mucosa via attaching and effacing (A/E) lesions. For years cultured cells were used as model systems to study A/E lesion formation, which showed actin accumulation under attached bacteria that can be raised above the plasma membrane in a pedestal-shaped structure. Studies of prototypical strains revealed that although both converge on N-WASP EPEC and EHEC O157:H7 use different actin polymerization pathways. While EPEC use the Tir-Nck pathway, Tir_EHECO157 cooperates with TccP/EspF_U to activate N-WASP. However, recent in vitro studies revealed a common EPEC and EHEC Tir-dependent and Nck-independent inefficient actin polymerization pathway. Unexpectedly, bacterial populations studies demonstrated that most non-O157 EHEC strains and EPEC lineage 2 strains can utilize both the Nck and TccP2 pathways in vitro . Importantly, in vivo and ex vivo mucosal infections have shown efficient A/E lesion formation independently of Nck and TccP. This review covers the progression in our understanding of EPEC and EHEC infection, through the different milestones obtained using cultured cells, to the realization that EPEC and EHEC have much more in common than previously appreciated and that mucosal attachment and microvillous effacement may be the key events, rather than pedestal formation.     

A tyrosine-phosphorylated 12-amino-acid sequence of enteropathogenic Escherichia coli Tir binds the host adaptor protein Nck and is required for Nck localization to actin pedestals

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) each promote the reorganization of actin into filamentous pedestal structures beneath attached bacteria during colonization of the intestinal epithelium. Central to this process is the translocation of the protein Tir (translocated intimin receptor) into the plasma membrane of host cells, where it interacts with the bacterial outer membrane protein intimin and triggers cellular signalling events that lead to actin rearrangement. Actin signalling by EPEC Tir requires a tyrosine residue, Y474, which is phosphorylated in the host cell. In contrast, EHEC Tir lacks this residue and generates pedestals independently of tyrosine phosphorylation. Consistent with this difference, recent work indicates that EHEC Tir cannot functionally replace EPEC Tir. To identify the role that tyrosine phosphorylation of EPEC Tir plays in actin signalling, we generated chimeric EHEC/EPEC Tir proteins and identified a 12-residue sequence of EPEC Tir containing Y474 that confers actin-signalling capabilities to EHEC Tir when the chimera is expressed in EPEC. Nck, a mammalian adaptor protein that has been implicated in the initiation of actin signalling, binds to this sequence in a Y474 phosphorylation-dependent manner and is recruited to the pedestals of EPEC, but not of EHEC.     

Host protein binding and adhesive properties of H6 and H7 flagella of attaching and effacing Escherichia coli

It had been suggested that the flagella of enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) might contribute to host colonization. In this study, we set out to investigate the adhesive properties of H7 and H6 flagella. We studied the abilities of EHEC EDL933 (O157:H7) and EPEC E2348/69 (O127:H6) flagella to bind to bovine mucus, host proteins such as mucins, and extracellular matrix proteins. Through several approaches, we found that H6 and H7 flagella and their flagellin monomers bind to mucins I and II and to freshly isolated bovine mucus. A genetic approach showed that EHEC and EPEC fliC deletion mutants were significantly less adherent to bovine intestinal tissue than the parental wild-type strains. In addition, we found that EPEC bacteria and H6 flagella, but not EHEC, bound largely, in a dose-dependent manner, to collagen and to a lesser extent to laminin and fibronectin. We also report that EHEC O157:H7 strains agglutinate rabbit red blood cells via their flagella, a heretofore unknown phenotype in this pathogroup. Collectively, our data demonstrate that the H6 and H7 flagella possess adhesive properties, particularly the ability to bind mucins, that may contribute to colonization of mucosal surfaces.     

Long-term Selenium Deficiency Increases the Pathogenicity of a Citrobacter rodentium Infection in Mice

Citrobacter rodentium is a mouse pathogen that causes infectious colitis and shares characteristics with human enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli, including the ability to cause attaching and effacing lesions in the colon and serves as a useful model to study the pathogenicity of these bacteria. In this study, mice were fed a selenium-deficient diet for 5 or 20?weeks and then infected with C. rodentium. Colonization of the colon by C. rodentium was similar in mice fed adequate or selenium-deficient diets, but total bacterial colonization of the spleen was elevated in mice fed selenium-deficient diet for 20?weeks. Infection-induced changes to the colon included inflammatory cell infiltration, gross changes in crypt architecture, and ulceration and denuding of the epithelial layer that were greatest in mice fed a selenium-deficient diet for 20?weeks. Expression of pro-inflammatory genes was significantly higher 12-days post-infection in mice fed the selenium-deficient diet for 20?weeks compared to mice fed a selenium-adequate diet or selenium-deficient diet for 5?weeks. Diarrhea was prevalent in mice fed the selenium-deficient diet for 20?weeks but not 5?weeks, and this was associated with decreased expression of solute carrier family 26a3 and carbonic anhydrase IV, genes involved in ion transport. These results indicated that selenium played an important role in resistance to the pathological effects of a C. rodentium infection, and therefore, selenium status may be important in the expression of human disease caused by common food-borne bacteria.     

Transient shielding of intimin and the type III secretion system of enterohemorrhagic and enteropathogenic Escherichia coli by a group 4 capsule

Enterohemorrhagic and enteropathogenic Escherichia coli (EHEC and EPEC, respectively) strains represent a major global health problem. Their virulence is mediated by the concerted activity of an array of virulence factors including toxins, a type III protein secretion system (TTSS), pili, and others. We previously showed that EPEC O127 forms a group 4 capsule (G4C), and in this report we show that EHEC O157 also produces a G4C, whose assembly is dependent on the etp, etk, and wzy genes. We further show that at early time points postinfection, these G4Cs appear to mask surface structures including intimin and the TTSS. This masking inhibited the attachment of EPEC and EHEC to tissue-cultured epithelial cells, diminished their capacity to induce the formation of actin pedestals, and attenuated TTSS-mediated protein translocation into host cells. Importantly, we found that Ler, a positive regulator of intimin and TTSS genes, represses the expression of the capsule-related genes, including etp and etk. Thus, the expression of TTSS and G4C is conversely regulated and capsule production is diminished upon TTSS expression. Indeed, at later time points postinfection, the diminishing capsule no longer interferes with the activities of intimin and the TTSS. Notably, by using the rabbit infant model, we found that the EHEC G4C is required for efficient colonization of the rabbit large intestine. Taken together, our results suggest that temporal expression of the capsule, which is coordinated with that of the TTSS, is required for optimal EHEC colonization of the host intestine.     

Citrobacter rodentium infection causes both mitochondrial dysfunction and intestinal epithelial barrier disruption in vivo: role of mitochondrial associated protein (Map)

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli are non-invasive attaching/effacing (A/E) bacterial pathogens that infect their host's intestinal epithelium, causing severe diarrhoeal disease. These bacteria utilize a type III secretion apparatus to deliver effector molecules into host cells, subverting cellular function. Mitochondrial associated protein (Map) is a multifunctional effector protein that targets host cell mitochondria and contributes to infection-induced epithelial barrier dysfunction in vitro. Unfortunately, the relevance of these actions to the pathogenesis of EPEC-induced disease is uncertain. Using Citrobacter rodentium, a mouse-adapted A/E bacterium, we found that Map colocalized with host cell mitochondria, and that in vivo infection led to a disruption of mitochondrial morphology in infected colonocytes as assessed by electron microscopy. Histochemical staining for the mitochondrial enzyme succinate dehydrogenase also revealed a significant loss of mitochondrial respiratory function in the infected intestinal epithelium; however, both pathologies were attenuated in mice infected with a Deltamap strain. C. rodentium Map was also implicated in the disruption of epithelial barrier function both in vitro and in vivo. These studies thus advance our understanding of how A/E pathogens subvert host cell functions and cause disease, demonstrating that Map contributes to the functional disruption of the intestinal epithelium during enteric infection by C. rodentium.     

Molecular evolution and mosaic structure of alpha, beta, and gamma intimins of pathogenic Escherichia coli.

Two types of pathogenic Escherichia coli, enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC), cause diarrheal disease by disrupting the intestinal environment through the intimate attachment of the bacteria to the intestinal epithelium. This process is mediated by intimin, an outer membrane protein that is homologous to the invasins of pathogenic Yersinia. The intimin (eae) gene is part of a pathogenicity island, a 35-kb segment of DNA that has been acquired independently in different groups of pathogens. Nucleotide sequences of eae of three EPEC and four EHEC strains representing distinct clonal lineages revealed an exceptionally high level of divergence (15%) in the amino acid sequences of alpha, beta, and gamma intimin molecules, most of which is concentrated in the C-terminal region. The gamma intimin sequences from E. coli strains with serotypes O157:H7, O55:H7, and O157:H- are virtually identical, supporting the hypothesis that these bacteria belong to a single clonal lineage. Sequences of beta intimin of EPEC strains of serotypes O111:H2 and O128:H2 show substantial differences from alpha and gamma intimins, indicating that these strains have evolved independently. Strong nonrandom clustering of polymorphic sites indicates that the intimin genes are mosaics, suggesting that protein divergence has been accelerated by recombination and diversifying selection.     

Enteropathogenic and enterohaemorrhagic Escherichia coli : more subversive elements

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.     

The EHEC type III effector NleL is an E3 ubiquitin ligase that modulates pedestal formation

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 causes hemorrhagic colitis and may result in potentially fatal hemolytic uremia syndrome in humans. EHEC colonize the intestinal mucosa and promote the formation of actin-rich pedestals via translocated type III effectors. Two EHEC type III secreted effectors, Tir and EspFu/TccP, are key players for pedestal formation. We discovered that an EHEC effector protein called Non-LEE-encoded Ligase (NleL) is an E3 ubiquitin ligase. In vitro, we showed that the NleL C753 residue is critical for its E3 ligase activity. Functionally, we demonstrated that NleL E3 ubiquitin ligase activity is involved in modulating Tir-mediated pedestal formation. Surprisingly, EHEC mutant strain deficient in the E3 ligase activity induced more pedestals than the wild-type strain. The canonical EPEC strain E2348/69 normally lacks the nleL gene, and the ectopic expression of the wild-type EHEC nleL, but not the catalytically-deficient nleL(C753A) mutant, in this strain resulted in fewer actin-rich pedestals. Furthermore, we showed that the C. rodentium NleL homolog is a E3 ubiquitin ligase and is required for efficient infection of murine colonic epithelial cells in vivo. In summary, our study demonstrated that EHEC utilizes NleL E3 ubiquitin ligase activity to modulate Tir-mediated pedestal formation.