Site-Specific Tyrosine Phosphorylation of the Type III Secretion Chaperone CesT Regulates Effector Hierarchy and Promotes Enteropathogenic Escherichia coli Intestinal Disease
Date
2017-08-22T15:25:37Z
Authors
Runte, Cameron
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Abstract
Enteropathogenic E. coli (EPEC) is a global enteric pathogen that causes serious gastrointestinal disease and inflicts a significant burden on healthcare systems worldwide. Recent phosphoproteomic studies suggest that phosphotyrosine modifications are widespread in bacteria and likely have significant impact on virulence factor regulation. In this study, we provide the first evidence that tyrosine phosphorylation of a multicargo chaperone, CesT, is functionally important for EPEC type III secretion system (T3SS)-
mediated pathogenesis. The conserved phosphosites, Y152 and Y153, are located within a unique domain of CesT at the C-terminus. Positional tyrosine to phenylalanine mutations resulted in differential secretion of CesT-dependent type III effectors, and loss of phenotypes associated with disease progression in vitro. These observations were validated in vivo using the closely related mouse pathogen Citrobacter rodentium, which requires CesT, and specifically a CesT phosphosite, for significant intestinal colonization. This study identifies a novel research direction to pursue limitation of EPEC pathogenesis.
Description
As a MSc. student at Dalhousie, I designed and implemented an interdisciplinary research project to study a particularly challenging protein domain suspected to be of critical importance to enteric E. coli pathogenesis. This project gave me a unique opportunity to design research that combined aspects of bacterial genetics, molecular biology, proteomics, microscopy, and infection biology.
The protein under study, CesT, is required for complete function of a type III secretion system (T3SS), which is a prokaryotic biological nanomachine that injects bacterial proteins directly into host cells in order to cause infection and disease. I established that a site-specific regulatory function exists for two neighbouring tyrosine phosphorylation modifications at the CesT C-terminal domain, and that CesT phosphorylation was required for specific molecular mechanisms critical to E. coli pathogenesis. Despite its expanded abundance of observation, a bacterial phosphorylation event critical to virulence is rare, and novel for the field of T3SS biology.
Several multidrug resistant bacterial strains are considered urgent threats by international organizations such as the United States Centres for Disease Control, and World Health Organizaiton. The research presented in this thesis identified a critical mechanism by which a bacterial pathogen regulates its virulence program, which is an important first step toward development of new pharmaceutical agents to limit this global enteric pathogen.
Keywords
Bacterial Genetics, Infection Biology, Mass Spectrometry, Fluorescence, Microscopy, Molecular Biology