UBC Theses and Dissertations
Elucidating the function of the type-III secreted effector proteins EspZ and NleC of the attaching and effacing bacterial pathogens Shames, Stephanie Rochelle
Enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC, respectively) are attaching and effacing (A/E) bacterial pathogens that cause diarrheal disease. EPEC causes severe infantile diarrhea in developing countries whereas EHEC infection results in severe bloody diarrhea worldwide. These pathogens employ a type-III secretion system (T3SS) encoded on the locus of enterocyte effacement (LEE) pathogenicity island (PAI) to inject a panel of effector proteins directly into infected host cells where they subvert host cell functions. The roles of many type-III secreted (T3S) effectors remain to be elucidated. Here, we have elucidated functions for the T3S effectors EspZ and NleC. EPEC infection causes host cell cytotoxicity and death. We demonstrated that EspZ enhances host cell survival during EPEC infection. Removal of espZ from the EPEC genome (∆espZ) exacerbated host cell cytotoxicity. We found that EspZ interacts with host CD98 and contributes to protection against EPEC-mediated cytotoxicity by enhancing phosphorylation of focal adhesion kinase (FAK). Further investigation revealed that EPEC ∆espZ infection caused a severe decrease in host mitochondrial inner membrane potential (∆ψm) concurrently with host cell lysis. We also found that EspZ localizes to host cell mitochondria and interacts with the translocase of inner mitochondrial membrane (TIM) 17B. These studies are the first to demonstrate EspZ function. Many non-LEE encoded (Nle) effector proteins impact innate immune signaling and we thus examined the contribution of the T3S zinc-metalloprotease NleC to this phenotype. We identified the host acetyltransferase p300 as a target of NleC and show that NleC causes decreased abundance of p300 in cellular nuclei. We further demonstrate that over-expression of p300 antagonizes repression of interleukin (IL)-8 secretion by EPEC and that small interfering ribonucleic acid (siRNA) knock-down of p300 dampens IL-8 secretion by EPEC ∆nleC-infected cells. Thesis work has identified a target of NleC and provided the first example of a bacterial virulence factor targeting the acetyltransferase p300. The work presented in this thesis provides novel insight into the function of two T3S effector proteins, EspZ and NleC. The mechanistic insight gained by these studies has thoroughly enhanced the understanding of these important virulence factors and their contribution to A/E pathogen infection.
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