Assembly of the Actin Pedestals Central to Adhesion of Enteropathogenic E. Coli

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. <P>
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 <OL> <LI> Cholesterol-rich membrane microdomains and receptor-kinase interaction<LI> How c-Fyn is recruited and regulated during pedestal formation<LI> How auxiliary cellular receptors might modulate Tir-dependent signalling. </ol> 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.