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.     

Enteropathogenic Escherichia coli (EPEC) attachment to epithelial cells: exploiting the host cell cytoskeleton from the outside

Enteropathogenic Escherichia coli (EPEC), a leading cause of human infantile diarrhoea, is the prototype for a family of intestinal bacterial pathogens that induce attaching and effacing (A/E) lesions on host cells. A/E lesions are characterized by localized effacement of the brush border of enterocytes, intimate bacterial attachment and pedestal formation beneath the adherent bacteria. As a result of some recent breakthrough discoveries, EPEC has now emerged as a fascinating paradigm for the study of host–pathogen interactions and cytoskeletal rearrangements that occur at the host cell membrane. EPEC uses a type III secretion machinery to attach to epithelial cells, translocating its own receptor for intimate attachment, Tir, into the host cell, which then binds to intimin on the bacterial surface. Studies of EPEC-induced cytoskeletal rearrangements have begun to provide clues as to the mechanisms used by this pathogen to subvert the host cell cytoskeleton and signalling pathways. These findings have unravelled new ways by which pathogenic bacteria exploit host processes from the cell surface and have shed new light on how EPEC might cause diarrhoea.     

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.     

Characterization of fimbriae produced by enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) express rope-like bundles of filaments, termed bundle-forming pili (BFP) (J. A. Girón, A. S. Y. Ho, and G. K. Schoolnik, Science 254:710-713, 1991). Expression of BFP is associated with localized adherence to HEp-2 cells and the presence of the EPEC adherence factor plasmid. In this study, we describe the identification of rod-like fimbriae and fibrillae expressed simultaneously on the bacterial surface of three prototype EPEC strains. Upon fimbrial extraction from EPEC B171 (O111:NM), three fimbrial subunits with masses of 16.5, 15.5, and 14.7 kDa were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Their N-terminal amino acid sequence showed homology with F9 and F7(2) fimbriae of uropathogenic E. coli and F1845 of diffuse-adhering E. coli, respectively. The mixture of fimbrial subunits (called FB171) exhibited mannose-resistant agglutination of human erythrocytes only, and this activity was not inhibited by alpha-D-Gal(1-4)-beta-Gal disaccharide or any other described receptor analogs for P, S, F, M, G, and Dr hemagglutinins of uropathogenic E. coli, which suggests a different receptor specificity. Hemagglutination was inhibited by extracellular matrix glycoproteins, i.e., collagen type IV, laminin, and fibronectin, and to a lesser extent by gangliosides, fetuin, and asialofetuin. Scanning electron microscopic studies performed on clusters of bacteria adhering to HEp-2 cells revealed the presence of structures resembling BFP and rod-like fimbriae linking bacteria to bacteria and bacteria to the eukaryotic cell membrane. We suggest a role of these surface appendages in the interaction of EPEC with eukaryotic cells as well as in the overall pathogenesis of intestinal disease caused by EPEC.     

A novel EspA-associated surface organelle of enteropathogenic Escherichia coli involved in protein translocation into epithelial cells.

Enteropathogenic Escherichia coli (EPEC), like many bacterial pathogens, employ a type III secretion system to deliver effector proteins across the bacterial cell. In EPEC, four proteins are known to be exported by a type III secretion system_EspA, EspB and EspD required for subversion of host cell signal transduction pathways and a translocated intimin receptor (Tir) protein (formerly Hp90) which is tyrosine-phosphorylated following transfer to the host cell to become a receptor for intimin-mediated intimate attachment and 'attaching and effacing' (A/E) lesion formation. The structural basis for protein translocation has yet to be fully elucidated for any type III secretion system. Here, we describe a novel EspA-containing filamentous organelle that is present on the bacterial surface during the early stage of A/E lesion formation, forms a physical bridge between the bacterium and the infected eukaryotic cell surface and is required for the translocation of EspB into infected epithelial cells.     

Enteropathogenic Escherichia coli inhibits ileal sodium-dependent bile acid transporter ASBT

Apical sodium-dependent bile acid transporter (ASBT) is responsible for the absorption of bile acids from the intestine. A decrease in ASBT function and expression has been implicated in diarrhea associated with intestinal inflammation. Whether infection with pathogenic microorganisms such as the enteropathogenic Escherichia coli (EPEC) affect ASBT activity is not known. EPEC is a food-borne enteric pathogen that translocates bacterial effector molecules via type three secretion system (TTSS) into host cells and is a major cause of infantile diarrhea. We investigated the effects of EPEC infection on ileal ASBT function utilizing human intestinal Caco2 cells and HEK-293 cells stably transfected with ASBT-V5 fusion protein (2BT cells). ASBT activity was significantly inhibited following 60 min infection with EPEC but not with nonpathogenic E. coli. Mutations in bacterial escN, espA, espB, and espD, the genes encoding for the elements of bacterial TTSS, ablated EPEC inhibitory effect on ASBT function. Furthermore, mutation in the bacterial BFP gene encoding for bundle-forming pili abrogated the inhibition of ASBT by EPEC, indicating the essential role for bacterial aggregation and the early attachment. The inhibition by EPEC was associated with a significant decrease in the V(max) of the transporter and a reduction in the level of ASBT on the plasma membrane. The inhibition of ASBT by EPEC was blocked in the presence of protein tyrosine phosphatase inhibitors. Our studies provide novel evidence for the alterations in the activity of ASBT by EPEC infection and suggest a possible effect for EPEC in influencing intestinal bile acid homeostasis.     

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.     

Targeting of enteropathogenic Escherichia coli EspF to host mitochondria is essential for bacterial pathogenesis: critical role of the 16th leucine residue in EspF

The attachment of enteropathogenic Escherichia coli (EPEC) to host cells and the induction of attaching and effacing (A/E) lesions are prominent pathogenic features. EPEC infection also leads to host cell death and damage to the intestinal mucosa, which is partly dependent upon EspF, one of the effectors. In this study, we demonstrate that EspF is a mitochondrial import protein with a functional mitochondrial targeting signal (MTS), because EspF activity for importing into the mitochondria was abrogated by MTS deletion mutants. Substitution of the 16th leucine with glutamic acid (EspF(L16E)) completely abolished EspF activity. Infection of HeLa cells with wild type but not the espF mutant (DeltaespF) decreased mitochondrial membrane potential (DeltaPsi(m)), leading to cell death. The DeltaPsi(m) decrease and cell death were restored in cells infected with DeltaespF/pEspF but not DeltaespF/pEspF(L16E), suggesting that the 16th leucine in the MTS is a critical amino acid for EspF function. To demonstrate the impact of EspF in vivo, we exploited Citrobacter rodentium by infecting C3H/HeJ mice with DeltaespF(CR), DeltaespF(CR)/pEspF(CR), or DeltaespF(CR)/pEspF(L16E)(CR). These results indicate that EspF activity contributes to bacterial pathogenesis, as judged by murine lethality and intestinal histopathology, and promotion of bacterial colonization of the intestinal mucosa.     

The ex vivo response of human intestinal mucosa to enteropathogenic Escherichia coli infection

In vitro organ culture (IVOC) represents a gold standard model to study enteropathogenic E. coli (EPEC) infection of human intestinal mucosa. However, the optimal examination of the bacterial–host cell interaction requires a directional epithelial exposure, without serosal or cut surface stimulation. A polarized IVOC system (pIVOC) was developed in order to overcome such limitations: apical EPEC infection produced negligible bacterial leakage via biopsy edges, resulted in enhanced colonization compared with standard IVOC, and showed evidence of bacterial detachment, as in natural rabbit EPEC infections. Examination of mucosal innate immune responses in pIVOC showed both interleukin (IL)-8 mRNA and protein levels were significantly increased after apical EPEC infection. Increased IL-8 levels mainly depended on flagellin expression as fliC -negative EPEC did not elicit a significant IL-8 response despite increased mucosal colonization compared with wild-type EPEC. In addition, apical application of purified flagella significantly increased IL-8 protein levels over non-infected controls. Immunofluorescence staining of EPEC-infected small intestinal biopsies revealed apical and basolateral distribution of Toll-like receptor (TLR) 5 on epithelium, suggesting that EPEC can trigger mucosal IL-8 responses by apical flagellin/TLR5 interaction ex vivo and does not require access to the basolateral membrane as postulated in cell culture models.     

A novel proline-rich protein, EspF, is secreted from enteropathogenic Escherichia coli via the type III export pathway

Enteropathogenic Escherichia coli (EPEC) cause a characteristic attaching and effacing (A/E) lesion in intestinal epithelial cells that is associated with the expression and export of specific bacterial proteins via a type III secretion pathway. These effector proteins and components of the type III export apparatus are encoded on a pathogenicity island known as the locus of enterocyte effacement (LEE). In this study, we describe a proline-rich protein, EspF, encoded by the LEE that is secreted by the EPEC type III secretion apparatus. Whereas an espF deletion mutant does not synthesize or secrete EspF, surprisingly it retains the ability to induce host signaling events, perform A/E activities, and invade host epithelial cells. Although these results do not indicate an obvious role for EspF in the formation of A/E lesions nor in the invasion of epithelial cells, they do not preclude a role played by EspF in other aspects of EPEC pathogenesis.     

Bundle-forming pilus retraction enhances enteropathogenic Escherichia coli infectivity

Enteropathogenic Escherichia coli (EPEC) is an important human pathogen that causes acute infantile diarrhea. The type IV bundle-forming pili (BFP) of typical EPEC strains are dynamic fibrillar organelles that can extend out and retract into the bacterium. The bfpF gene encodes for BfpF, a protein that promotes pili retraction. The BFP are involved in bacterial autoaggregation and in mediating the initial adherence of the bacterium with its host cell. Importantly, BFP retraction is implicated in virulence in experimental human infection. How pili retraction contributes to EPEC pathogenesis at the cellular level remains largely obscure, however. In this study, an effort has been made to address this question using engineered EPEC strains with induced BFP retraction capacity. We show that the retraction is important for tight-junction disruption and, to a lesser extent, actin-rich pedestal formation by promoting efficient translocation of bacterial protein effectors into the host cells. A model is proposed whereby BFP retraction permits closer apposition between the bacterial and the host cell surfaces, thus enabling timely and effective introduction of bacterial effectors into the host cell via the type III secretion apparatus. Our studies hence suggest novel insights into the involvement of pili retraction in EPEC pathogenesis.     

Genetically engineered enteropathogenic Escherichia coli strain elicits a specific immune response and protects against a virulent challenge

Enteropathogenic Escherichia coli (EPEC), a major cause of severe disease with diarrhea in infants, is also involved in weaned rabbit colibacillosis. EPEC O103 is frequent in rabbit-fattening units of Western Europe. It causes high mortality and growth retardation, leading to substantial economic losses. We report here the construction by allelic exchange of an EPEC O103 strain mutated in espB and tir, two essential virulence genes. Upon live oral administration to weaned rabbits, the E22DeltaTir/EspB mutant strain efficiently colonized the intestinal tract without any adverse consequences. The rabbits were challenged with the highly pathogenic parental strain E22. The mutant provided complete protection to rabbits and total resistance to intestinal colonization by E22. In addition, E22DeltaTir/EspB strain induced a specific humoral response against the bacterial adhesin AF/R2. These Abs prevent bacterial attachment to epithelial cells in vitro. These results open the way for the development of an efficient vaccine strategy against rabbit EPEC infections.     

Proteomic analysis of the binding partners to enteropathogenic Escherichia coli virulence proteins expressed in Saccharomyces cerevisiae

Enteropathogenic Escherichia coli (EPEC) is an enteric human pathogen responsible for much worldwide morbidity and mortality. EPEC uses a type III secretion system to inject bacterial proteins into the cytosol of intestinal epithelial cells to cause diarrheal disease. We are interested in determining the host proteins to which EPEC translocator and effector proteins bind during infection. To facilitate protein enrichment, we created fusions between GST and EPEC virulence proteins, and expressed these fusions individually in Saccharomyces cerevisiae. The biology of S. cerevisiae is well understood and often employed as a model eukaryote to study the function of bacterial virulence factors. We isolated the yeast proteins that interact with individual EPEC proteins by affinity purifying against the GST tag. These complexes were subjected to ICAT combined with ESI-MS/MS. Database searching of sequenced peptides provided a list of proteins that bound specifically to each EPEC virulence protein. The dataset suggests several potential mammalian targets of these proteins that may guide future experimentation.     

Infection of rabbit kidney cells (RK13) by enteropathogenic Escherichia coli as a model to study the dynamics of actin cytoskeleton

Enteropathogenic Escherichia coli (EPEC) colonizes the intestinal mucosa and causes a cell lesion known as attachment and effacement (A/E) lesion. The molecular mechanisms for A/E lesions include injection of Tir, which is a receptor for an adhesin named intimin. The Tir-intimin interaction causes rearrangement of the cytoskeleton forming actin-rich structures called pedestals. Unfortunately, the formation of the A/E lesions and the dynamics of the actin cytoskeleton during this rearrangement induced by EPEC cannot be studied in the natural host. However, there are EPEC strains that infect rabbit (REPEC) that are genetically and pathologically similar to EPEC. Here, we used REPEC for the infection of rabbit kidney epithelial cells, line RK13, as a model to understand the actin cytoskeleton dynamics during pedestal formation. Actin-rich pedestal formation during the infection of RK13 cells by REPEC was analyzed by electron and confocal microscopy. The kinetics of infection along with the use of antibiotics for eliminating the bacteria, as well as reinfection, evidenced the plasticity of the actin cytoskeleton during pedestal formation. Thus, this model is a helpful tool for studying the dynamics of actin cytoskeleton and for correlating the data with those observed in in vivo models in rabbits experimentally infected with REPEC.     

Esp-independent functional integration of the translocated intimin receptor (Tir) of enteropathogenic Escherichia coli (EPEC) into host cell membranes

The pathogenesis of enteropathogenic Escherichia coli (EPEC) is characterized by the type III secretion system-dependent exploitation of target cells that results in attaching and effacing (A/E) lesions, actin rearrangements and pedestal formation. This pathology is mediated by effector proteins which are translocated by the type III secretion system into the host cell such as the translocated intimin receptor (Tir) and several E. coli secreted proteins (Esp). Secretion of virulence proteins of EPEC is tightly regulated. In response to Ca(2+), Esp secretion is drastically reduced, whereas secretion of Tir is increased. Membrane insertion of Tir, secreted under low Ca(2+) conditions, is therefore independent of Esp. Furthermore, espB and espD mutant strains of EPEC, unable to form the translocation pore, still translocate Tir into host cells membranes. This autointegrated Tir is functional, as it is able to complement a tir mutant strain in recruiting actin to bacterial contact sites. The uptake of Tir into the host cell appears to depend on the C-terminal part of the protein, as deletion of this part of Tir prevents autointegration. Together, our results demonstrate that under conditions of limited Ca(2+) an alternative mechanism for Tir integration can trigger the induction of A/E lesions.     

Attaching and effacing pathogen-induced tight junction disruption in vivo

Diarrhoea is a hallmark of infections by the human attaching and effacing (A/E) pathogens, enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). Although the mechanisms underlying diarrhoea induced by these pathogens remain unknown, cell culture results have suggested that these pathogens may target tight junctions. Tight junctions in the colon function as physical intercellular barriers that separate and prevent mixing of the luminal contents with adlumenal regions of the epithelium. Consequently, it is thought that the disruption of intestinal epithelial tight junctions by A/E pathogens could result in a loss of barrier function in the alimentary tract; however, this remains unexamined. Here we demonstrate for the first time that A/E pathogen infection results in the morphological alteration of tight junctions during natural disease. Tight junction alteration, characterized by relocalization of the transmembrane tight junction proteins claudin 1, 3 and 5, is a functional disruption; molecular tracers, which do not normally penetrate uninfected epithelia, pass across pathogen-infected epithelia. Functional junction disruption occurs with a concomitant increase in colon luminal water content. The effects on tissue are dependent upon the bacterial type III effector EspF (E. coli secreted protein F), because bacteria lacking EspF, while able to colonize, are defective for junction disruption and result in decreased proportions of water in the colon compared with wild-type infection. These results suggest that the diarrhoea induced by A/E pathogens occurs as part of functional tight junction disruption.     

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.     

The coiled-coil domain of EspA is essential for the assembly of the type III secretion translocon on the surface of enteropathogenic Escherichia coli

Enteropathogenic E. coli (EPEC) utilize a type III secretion system to deliver virulence-associated effector proteins to the host cell. Four proteins, EspA, EspB, EspD, and Tir, which are integral to the formation of characteristic "attaching and effacing" (A/E) intestinal lesions, are known to be exported via the EPEC type III secretion system. Recent work demonstrated that EspA is a major component of a filamentous structure, elaborated on the surface of EPEC, which is required for translocation of EspB and Tir. The carboxyl terminus of EspA is predicted to comprise an alpha-helical region, which demonstrates heptad periodicity whereby positions a and d in the heptad repeat unit abcdefg are occupied by hydrophobic residues, indicating a propensity for coiled-coil interactions. Here we demonstrate multimeric EspA isoforms in EPEC culture supernatants and EspA:EspA interaction on solid phase. Non-conservative amino acid substitution of specific EspA heptad residues generated EPEC mutants defective in filament assembly but which retained the ability to induce A/E lesions; additional mutation totally abolished EspA filament assembly and A/E lesion formation. These results demonstrate a similarity to flagellar biosynthesis and indicate that the coiled-coil domain of EspA is required for assembly of the EspA filament-associated type III secretion translocon.     

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.