Investigating the molecular mechanisms of enteropathogenic Escherichia coli pathogenesis
AuthorPollock, Georgina Lauren
AffiliationMicrobiology & Immunology
Document TypePhD thesis
Access StatusThis item is embargoed and will be available on 2021-09-26.
© 2019 Georgina Lauren Pollock
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. Once inside the host cell, these effector proteins modulate normal host cell processes, such as cytoskeletal dynamics and cell autonomous defenses, to aid in bacterial pathogenesis. One such effector protein is the Non-LEE encoded effector NleB1, which inhibits extrinsic apoptotic signaling via the FAS death receptor. NleB1 transfers a single GlcNAc residue to Arg117 in the death domain of FADD and inhibits FAS ligand (FasL) stimulated caspase-8 cleavage. Another effector secreted by the T3SS is NleF. Previous studies have shown that NleF binds to and inhibits the activity of caspase-4, -8 and -9 in vitro. Here we investigated a role for NleF in the inhibition of FAS signaling and apoptosis during EPEC infection. We show NleF prevents the cleavage of caspase-8, caspase-3 and RIPK1 in response to FasL stimulation. When translocated into host cells by the T3SS or expressed ectopically, NleF also blocked FasL-induced cell death. Using the EPEC-like mouse pathogen C. rodentium we found that NleB but not NleF contributed to colonization of mice in the intestine. Hence, despite their shared ability to block FasL/FAS signaling, NleB and NleF appear to have distinct roles during infection. Another two translocated effectors are NleH1 and NleH2, which together form a new family of bacterial kinases. The phosphorylated targets of these kinases within the host cell have not been previously described. 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 protein interaction analysis 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. We examined 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.
Keywordspathogenic E. coli; EPEC; apoptosis; caspase inhibitor; FAS signalling; bacterial kinase; AE lesion; microvilli effacement
- Click on "Export Reference in RIS Format" and choose "open with... Endnote".
- Click on "Export Reference in RIS Format". Login to Refworks, go to References => Import References