Assembly of the actin pedestals central to adhesion of enteropathogenic E.coli

Disease-causing (pathogenic) bacteria are a major health problem for man and animals, and the severe threat they pose has become a public concern as problems arise for example in hospitals and the food sector, and evidence is reported of the seemingly inexorable increase in antibiotic resistance. Food-borne diarrhoeal disease caused by enteropathogenic E.coli (EPEC) and its relative enterohemorrhagic E.coli O157:H7 (EHEC) are a serious burden to economically developed countries and the Third World. After ingestion EPEC and EHEC must first stick tightly to cells of the intestine, and to do this the bacteria deliver a harpoon-like protein (Tir) into the human cell membrane which they bind to using a second protein (intimin) fixed on the bacterial surface. This Velcro-like interaction induces the cell to assemble a tower-like pedestal to which the bacteria are tightly bound. It has been known for about 5 years that EPEC Tir protein is modified when it is inserted into the human cell membrane, and that this modification is the cue that is key to inducing host responses, in particular triggering reorganisation of the internal cell skeleton needed to build the pedestal .For the last 3 years, we have used biochemical and cell biology approaches to investigate how intimin and Tir work. We have been able to demonstrate that intimin-Tir binding induces Tir modification, and have identified the host cellular protein called Fyn that controls this critical event. We also showed that cholesterol in the host target membrane is important for delivery of Tir and for its activity. We propose to study further the central role of the host Fyn-dependent mechanism of EPEC pedestal assembly. The resulting information will give us a clearer view of this critical early step in pathogen-host interaction and provide information of potential importance in developing new tests and drugs to combat infection. Studying Tir also tells us more about how such cell membrane receptor proteins work, also an important area of research as they normally act to control processes like development, growth and our immune system, and when they malfunction are involved in diseases like cancer.

Diarrhoeal disease caused by enterobacteria such as enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E.coli is a major human health problem worldwide, accounting for substantial morbidity and mortality. The control of these pathogens is threatened by the emergence of antibiotic resistance and the paucity of effective vaccines. An understanding of the molecular components underlying how they establish infections is therefore important. EPEC and EHEC initiate infection by tightly attaching to human and animal intestinal cells. To do this they trigger reorganisation of the host cellular actin cytoskeleton, resulting in the assembly of ?pedestal-like? pseudopods binding the bacteria to the host cell surface. The key event of pedestal assembly is induced by two bacterial effector proteins that mimic a ligand-receptor interaction ? the translocated intimin receptor (Tir) that the bacteria deliver into the host cell plasma membrane, and the bacterial surface protein intimin, which engages Tir. EPEC Tir is phosphorylated on tyrosine 474 (Y474), and binds the cellular adaptor Nck, which recruits N-WASP and Arp2/3 to trigger actin polymerisation. In 2002, we initiated a pilot study to understand the central features of the intact EPEC outer membrane protein intimin and its interaction with Tir. We showed that binding of dimeric EPEC intimin induces Tir clustering and Y474 phosphorylation. Using pharmacological inhibition, knockout cells, in vitro assays and siRNA knockdown, we demonstrated that the cellular Src-family kinase (SFK) c-Fyn phosphorylates Tir Y474 downstream of intimin-directed receptor clustering, a signal essential for Nck-actin pedestal assembly by EPEC Tir. We also showed a requirement for cellular cholesterol both in Tir delivery and downstream signalling. We propose to establish a funded Project to build on these findings, in particular to study further the central role of c-Fyn in actin pedestal assembly by EPEC. Specifically, we will continue to use a combination of biochemical and cell biology approaches to analyse 1) cholesterol-rich membrane microdomains and receptor-kinase interaction, 2) how c-Fyn is recruited and regulated during pedestal formation, 3) how auxiliary cellular receptors might modulate Tir-dependent signalling. Understanding c-Fyn dependent Tir phosphorylation in detail will yield important new insights into the molecular mechanisms underlying pedestal formation and EPEC pathogenesis, and illuminate how eukaryotic kinases recognise receptors.