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Santos, Andrew Stanislaw

University of British Columbia

Enteropathogenic Escherichia coli (EPEC) is a bacterial pathogen that causes significant morbidity and mortality worldwide. During infection, EPEC attaches to the intestinal epithelium and uses a molecular syringe-like nanomachine called a Type 3 Secretion system (T3SS) to secrete virulence ‘effector’ proteins directly into human cells, where they manipulate host cell signalling and mediate the characteristic features of infection, including intimate attachment to host cells, formation of actin-rich pedestals, disruption of the epithelial barrier, and dysregulated immune function and cell survival. Some effectors possess enzymatic activities that allow them to directly modify host proteins through post-translational modifications, including ubiquitination, phosphorylation, and proteolysis. However, the host targets of many effectors are unknown and their collective impact on global host post-translational modification has not been widely explored. This work utilizes proteomics tools to characterize the impact of EPEC infection and T3SS effectors on the host proteome. Here, ubiquitin proteomics were used to investigate the EPEC effector ubiquitin ligase NleG, exploring its impact on the host ubiquitome, and identifying and characterizing the relationship of NleG with a novel host substrate: Ataxin-3. We also developed a novel EPEC infection-proteomics workflow to characterize global changes in host protein post-translational modifications during important stages of infection. This method simultaneously profiled changes in protein abundance, ubiquitination, and phosphorylation due to EPEC infection, T3SS effector influences, and host response during early, mid-, and late infection. Infection-proteomics exhibited the highest coverage of the host proteome, ubiquitome, and phosphoproteome of any EPEC proteomics investigation to date, identifying thousands of EPEC infection-, exposure-, and T3SS-associated changes in proteins involved in cell processes regulated by EPEC during infection. These include innate immune signalling, actin remodelling, and cell survival pathways, while also identifying novel pathways targeted by EPEC. This work demonstrates the power of proteomics techniques to characterize impacts of EPEC infection on the host proteome by developing a novel approach to study how EPEC infection and effectors affect host post-translational modification and cell signalling throughout infection. Proteomics tools serve as valuable methods to study host-pathogen interactions and identifying molecular level changes underpinning characteristic phenotypes mediated by EPEC during infection.

Text

2024-01-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/83601

Microbiology and Immunology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/