Brendan Kenny

ENTEROPATHOGENIC E. COLI (EPEC) TRANSFERS ITS RECEPTOR FOR INTIMATE ADHERENCE INTO MAMMALIAN CELLS

Enteropathogenic E. coli (EPEC) belongs to a group of bacterial pathogens that induce epithelial cell actin rearrangements resulting in pedestal formation beneath adherent bacteria. This requires the secretion of specific virulence proteins needed for signal transduction and intimate adherence. EPEC interaction induces tyrosine phosphorylation of a protein in the host membrane, Hp90, which is the receptor for the EPEC outer membrane protein, intimin. Hp90-intimin interaction is essential for intimate attachment and pedestal formation. Here, we demonstrate that Hp90 is actually a bacterial protein (Tir). Thus, this bacterial pathogen inserts its own receptor into mammalian cell surfaces, to which it then adheres to trigger additional host signaling events and actin nucleation. It is also tyrosine-phosphorylated upon transfer into the host cell.

EspA, a protein secreted by enteropathogenic Escherichia coli, is required to induce signals in epithelial cells

Enteropathogenic Escherichia coli (EPEC) is a leading cause of infant diarrhoea. EPEC mediates several effects on host epithelial cells, including activation of signal‐transduction pathways, cytoskeletal rearrangement along with pedestal and attachingleffacing lesion formation. It has been previously shown that the EPEC eaeB (espB) gene encodes a secreted protein required for signal transduction and adherence, while eaeA encodes intimin, an EPEC membrane protein that mediates intimate adherence and contributes to focusing of cytoskeletal proteins beneath bacteria. DNA‐sequence analysis of a region between eaeA and eaeB identified a predicted open reading frame (espA) that matched the amino‐terminal sequence of a 25 kDa EPEC secreted protein. A mutant with a non‐polar insertion in espA does not secrete this protein, activate epithelial cell signal transduction or cause cytoskeletal rearrangement. These phenotypes were complemented by a cloned espA gene. The espA mutant is also defective for invasion. It is concluded that espA encodes an EPEC secreted protein that is necessary for activating epithelial signal transduction, intimate contact, and formation of attaching and effacing lesions, processes which are central to pathogenesis.

Complete Genome Sequence and Comparative Genome Analysis of Enteropathogenic Escherichia coli O127:H6 Strain E2348/69

Enteropathogenic Escherichia coli (EPEC) was the first pathovar of E. coli to be implicated in human disease; however, no EPEC strain has been fully sequenced until now. Strain E2348/69 (serotype O127:H6 belonging to E. coli phylogroup B2) has been used worldwide as a prototype strain to study EPEC biology, genetics, and virulence. Studies of E2348/69 led to the discovery of the locus of enterocyte effacement-encoded type III secretion system (T3SS) and its cognate effectors, which play a vital role in attaching and effacing lesion formation on gut epithelial cells. In this study, we determined the complete genomic sequence of E2348/69 and performed genomic comparisons with other important E. coli strains. We identified 424 E2348/69-specific genes, most of which are carried on mobile genetic elements, and a number of genetic traits specifically conserved in phylogroup B2 strains irrespective of their pathotypes, including the absence of the ETT2-related T3SS, which is present in E. coli strains belonging to all other phylogroups. The genome analysis revealed the entire gene repertoire related to E2348/69 virulence. Interestingly, E2348/69 contains only 21 intact T3SS effector genes, all of which are carried on prophages and integrative elements, compared to over 50 effector genes in enterohemorrhagic E. coli O157. As E2348/69 is the most-studied pathogenic E. coli strain, this study provides a genomic context for the vast amount of existing experimental data. The unexpected simplicity of the E2348/69 T3SS provides the first opportunity to fully dissect the entire virulence strategy of attaching and effacing pathogens in the genomic context.

Targeting of an enteropathogenic Escherichia coli (EPEC) effector protein to host mitochondria

Many Gram‐negative pathogens use a type III secretion apparatus to deliver effector molecules into host cells to subvert cellular processes in favour of the pathogen. Enteropathogenic Escherichia coli (EPEC) uses such a system to deliver the Tir effector molecule into host cells. In this paper, we show that the gene upstream of tir, orf19, encodes an additional type III secreted effector protein. Orf19 is delivered into host cells by a mechanism independent of endocytosis, but dependent on EspB. Orf19 is targeted to host mitochondria, where it appears to interfere with the ability to maintain membrane potential. Although the precise role of Orf19 remains to be elucidated, its interaction with mitochondria suggests a possible role in the subversion of key functions of these organelles, such as energy production or control of cell death. This is the first example of a type III secreted protein targeted to mitochondria; it is probable that homologues (present in EPEC and Shigella species) and other bacterial effectors will also target this organelle.

Phosphorylation of tyrosine 474 of the enteropathogenic Escherichia coli (EPEC) Tir receptor molecule is essential for actin nucleating activity and is preceded by additional host modifications.

The enteropathogenic Escherichia coli (EPEC) Tir protein becomes tyrosine phosphorylated in host cells and displays an increase in apparent molecular mass. The interaction of Tir with the EPEC outer membrane protein, intimin, triggers actin nucleation beneath the adherent bacteria. The enterohaemorrhagic E. coli 0157:H7 (EHEC) Tir molecule is not tyrosine phosphorylated. In this paper, Tir tyrosine phosphorylation is shown to be essential for actin nucleation activity, but not for the increase in apparent molecular mass observed in target cells. Tyrosine phosphorylation had no role in Tir molecular mass shift, indicating additional host modifications. Analysis of Tir intermediates indicates that tyrosine‐independent modification functions to direct Tirs correct insertion from the cytoplasm into the host membrane. Deletion analysis identified Tir domains participating in translocation, association with the host membrane, modification and antibody recognition. Intimin was found to bind a 55‐amino‐acid region (TIBA) within Tir that topological and sequence analysis suggests is located in an extracellular loop. Homologous TIBA sequences exist in integrins, which also bind intimin. Collectively, this study provides definitive evidence for the importance of tyrosine phosphorylation for EPEC Tir function and reveals differences in the pathogenicity of EPEC and EHEC. The data also suggest a mechanism for Tir insertion into the host membrane, as well as providing clues to the mode of intimin–integrin interaction.

IpaB mediates macrophage apoptosis induced by Shigella flexneri

Shigella flexneri kills macrophages through apoptosis, involving the induction of host cell DNA fragmentation and characteristic morphological changes. Shigella can only cause damage if it escapes from the phagolysosome into the cytoplasm. The S. flexneri cytotoxic genes have been localized to the ipa operon of shigellas virulence plasmid. ipaB, C and D deletion mutants are not invasive and therefore not cytotoxic. In order to distinguish genes involved in the escape from the phagolysosome as distinct from cytotoxicity, we constructed Shigella strains that secrete low amounts of Escherichia coli haemolysin (hlylow). These strains can escape into the cytoplasm of the macrophage even in the absence of the invasion plasmid as verified by electron microscopy and resistance to chloroquine. Macrophages were infected with different ipa mutants expressing hlylow. Both δipaC hlylow and δipaD hlylow were cytotoxic whilst δipaB hlylow and a hlylow strain cured of shigellas pathogenicity plasmid were not. Furthermore, both δipC ahlylow and δipaD hlylow killed through apoptosis as shown by both changes in ultrastructural morphology and fragmentation of the host ceil DNA. These results demonstrate that ipaB is essential for S. flexneri to induce apoptosis in macrophages.

Co-ordinate regulation of distinct host cell signalling pathways by multifunctional enteropathogenic Escherichia coli effector molecules

Enteropathogenic Escherichia coli (EPEC) is a major cause of paediatric diarrhoea and a model for the family of attaching and effacing (A/E) pathogens. A/E pathogens encode a type III secretion system to transfer effector proteins into host cells. The EPEC Tir effector protein acts as a receptor for the bacterial surface protein intimin and is involved in the formation of Cdc42‐independent, actin‐rich pedestal structures beneath the adhered bacteria. In this paper, we demonstrate that EPEC binding to HeLa cells also induces Tir‐independent, cytoskeletal rearrangement evidenced by the early, transient formation of filopodia‐like structures at sites of infection. Filopodia formation is dependent on expression of the EPEC Map effector molecule – a protein that targets mitochondria and induces their dysfunction. We show that Map‐induced filopodia formation is independent of mitochondrial targeting and is abolished by cellular expression of the Cdc42 inhibitory WASP‐CRIB domain, demonstrating that Map has at least two distinct functions in host cells. The transient nature of the filopodia is related to an ability of EPEC to downregulate Map‐induced cell signalling that, like pedestal formation, was dependent on both Tir and intimin proteins. The ability of Tir to downregulate filopodia was impaired by disrupting a putative GTPase‐activating protein (GAP) motif, suggesting that Tir may possess such a function, with its interaction with intimin triggering this activity. Furthermore, we also found that Map‐induced cell signalling inhibits pedestal formation, revealing that the cellular effects of Tir and Map must be co‐ordinately regu‐lated during infection. Possible implications of the multifunctional nature of EPEC effector molecules in pathogenesis are discussed.

The effector repertoire of enteropathogenic E. coli: ganging up on the host cell.

Diarrhoeal disease caused by enteropathogenic E. coli (EPEC) is dependent on a delivery system that injects numerous bacterial ‘effector’ proteins directly into host cells. The best-described EPEC effectors are encoded together on the locus of enterocyte effacement (LEE) pathogenicity island and display high levels of multifunctionality and cooperativity within the host cell. More recently, effectors encoded outside the LEE (non-LEE effectors) have been discovered and their functions are beginning to be uncovered. The recent completion of the EPEC genome sequence suggests its effector repertoire consists of at least 21 effector proteins. Here, we describe the genomic location of effectors and discuss recent advances made on effector cellular function as well as their role in the infection process.

Intestinal barrier dysfunction by enteropathogenic Escherichia coli is mediated by two effector molecules and a bacterial surface protein

The human intestinal pathogen, enteropathogenic Escherichia coli (EPEC), causes diarrhoeal disease by a mechanism that is dependent on the injection of effector proteins into the host cell. One effector, EspF, is reported to be required for EPEC to disrupt tight junction integrity of intestinal cells and increase the paracellular movement of molecules, which is likely to  contribute  to  diarrhoea.  Here,  we  show  that  not one but three EPEC‐encoded factors play important roles in this process. Thus, the Map (Mitochondria‐associated protein) effector is shown to: (i) be as essential as EspF for disrupting intestinal barrier function, (ii) be able to function independently of EspF, (iii) alter tight junction structure and (iv) mediate these effects in the absence of mitochondrial targeting. Additionally, the outer membrane protein Intimin is shown to be crucial for EspF and Map to disrupt the intestinal barrier function. This function of Intimin is completely independent of its interaction with its known receptor Tir, revealing a physiologically relevant requirement for Intimin interaction with alternative receptor(s). This work demonstrates that EPEC uses multiple multifunctional proteins to elicit specific responses in intestinal cells and that EPEC can control the activity of its injected effector molecules from its extracellular location.

Enteropathogenic Escherichia coli (EPEC) inactivate innate immune responses prior to compromising epithelial barrier function.

Enteropathogenic Escherichia coli (EPEC) infection of the human small intestine induces severe watery diarrhoea linked to a rather weak inflammatory response despite EPECs in vivo capacity to disrupt epithelial barrier function. Here, we demonstrate that EPEC flagellin triggers the secretion of the pro‐inflammatory cytokine, interleukin (IL)‐8, from small (Caco‐2) and large (T84) intestinal epithelia model systems. Interestingly, IL‐8 secretion required basolateral infection of T84 cells implying that flagellin must penetrate the epithelial barrier. In contrast, apical infection of Caco‐2 cells induced IL‐8 secretion but less potently than basolateral infections. Importantly, infection of Caco‐2, but not T84 cells rapidly inhibited IL‐8 secretion by a mechanism dependent on the delivery of effectors through a translocation system encoded on the locus of enterocyte effacement (LEE). Moreover, EPEC prevents the phosphorylation‐associated activation of multiple kinase pathways regulating IL‐8 gene transcription by a mechanism apparently independent of LEE‐encoded effectors and four non‐LEE‐encoded effectors. Crucially, our studies reveal that EPEC inhibits the capacity of the cells to secrete IL‐8 in response to bacterial antigens and inflammatory cytokines prior to disrupting barrier function by a distinct mechanism. Thus, these findings also lend themselves to a plausible mechanism to explain the absence of a strong inflammatory response in EPEC‐infected humans.