During infection the gastrointestinal pathogen enteropathogenic Escherichia coli (EPEC) forms a characteristic lesion on the surface of infected enterocytes known as an attaching/effacing lesion. Defining features of this lesion include intimate attachment of the bacteria to the host surface, manipulation of the host cytoskeleton beneath the site of bacterial attachment, and effacement of the surrounding microvilli. EPEC utilises a type III secretion system (T3SS) to translocate effector proteins directly into the cytosol of infected cells. Two translocated effectors are NleH1 and NleH2, which together form a new family of bacterial kinases. The target of these kinases within the host is currently unknown.
To identify putative host targets of NleH1 and NleH2 kinase activity, we conducted a phospho-proteomic comparison of intestinal epithelial cells infected with wild type EPEC or an EPEC double mutant lacking nleH1 and nleH2. We identified previously undescribed serine phosphorylation of the host proteins Eps8, Eps8L1 and Eps8L2 during wild type infection, that was absent during infection with EPEC ΔnleH1 ΔnleH2. Phosphorylation of Eps8 by NleH1 and NleH2 kinases was confirmed in vitro, in addition we used co-immunoprecipitation and yeast-2-hybrid to demonstrate an interaction between Eps8 and the bacterial kinases.
Eps8 is a dual function actin capping and bundling protein that specifically localises to the distal tips of microvilli. Loss of Eps8 results in microvilli shortening in mouse intestinal models and polarized epithelial cells. Current work is focusing on dissecting the impact of phosphorylation on Eps8 function and investigating a role for NleH1 and NleH2 in the microvilli effacement observed during EPEC infection. This work extends our understanding of defensive mechanisms in the intestinal brush border and how pathogenic bacteria overcome these to cause disease.