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, effacement of the surrounding microvilli and manipulation of the host cytoskeleton beneath the site of bacterial attachment. 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 form a new family of bacterial kinases, however, the targets of these kinases within the host remains unclear.
To identify putative host targets of NleH1 and NleH2, we conducted a phosphoproteomic comparison of cells infected with wild type EPEC with cells infected with an EPEC double mutant lacking nleH1 and nleH2. We observed a previously undescribed serine phosphorylation event of the host proteins Eps8, Eps8L1 and Eps8L2 during wild type infection, but not during infection with EPEC ΔnleH1 ΔnleH2. Phosphorylation of Eps8L1 was also observed during ectopic expression of NleH1, but not during expression of a catalytically inactive version of the kinase, NleH1K159A.
The Eps8 family possess actin bundling activity and are essential for microvilli formation. We demonstrated ectopic expression of NleH1 or NleH2 disrupted the actin bundling activity of Eps8, as did introducing a phosphomimetic mutation to Eps8. Further, interaction of NleH1 and NleH2 with Eps8 was confirmed with co-immunoprecipitation and yeast-2-hybrid studies.
Future work will dissect the impact of phosphorylation on Eps8 function, investigate the role of Eps8 in EPEC attaching/effacing lesion formation, and may explain the effect of EPEC infection on microvillus length and function.