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.

SummaryThe 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 Tir's 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.

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. Escherichia coli is important because it is biology's premier model organism, is a common commensal of the vertebrate gut, and is a versatile pathogen of humans and animals. Molecular epidemiological studies have classified E. coli strains into a number of phylogroups (phylogroups A, B1, B2, D, and E) (13, 42), which are estimated to have diverged in the last 5 to 9 million years (37, 42). Commensal E. coli strains are beneficial to the host and rarely cause disease. However, several clones of E. coli are responsible for a spectrum of diseases, including urinary tract infection, sepsis/meningitis, and diarrhea (for a review, see reference 15). Diarrheagenic E. coli strains are divided into enterotoxigenic E. coli (ETEC), en-

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.

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 attaching/effacing 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.