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Krekhno, Zakhar

University of British Columbia

Infectious diarrheal diseases are the third leading cause of mortality in young children, many of which are driven by Gram-negative bacterial pathogens, including enteropathogenic Escherichia coli (EPEC). This thesis employed an amalgamation of omics, in silico prediction, and conventional molecular biology tools to investigate the host-pathogen interactions during EPEC infection, primarily focusing on the systems-level and protein interactions. We employed a dual RNA-sequencing approach to investigate the host and microbe physiology during EPEC infection of an intestinal epithelial cell line (Caco-2/TC-7) with a focus on the role of the Type III Secretion System (T3SS). Our findings showed that T3SS was used by EPEC to suppress various host responses, including immune signaling and apoptosis. We hypothesized that microRNAs (miRNAs) were important for mediating host and pathogen behaviors and identified differentially expressed miRNAs that played a role in suppressing cell death and altering cytokine secretion in response to infection. With regards to bacterial transcriptome, we observed drastic upregulation of the Type II Secretion System (T2SS) genes in EPEC upon host cell contact. To explore the role of the T2SS during natural host infection, we used Citrobacter rodentium, a murine enteric pathogen, as a model of EPEC-caused disease. We demonstrated that this system was functional in vitro with potential roles in intestinal mucin degradation. During host infection, loss of the T2SS or predicted effectors led to a significant colonization defect and lack of systemic spread. Finally, apoptosis was among the pathways with a T3SS-associated pattern of dysregulation during infection. This prompted us to investigate the function of a pro-apoptotic T3SS effector, Map, due to the lack of understanding of its mechanism of action. Through a combination of proximity labelling mass spectrometry and deep learning interaction prediction algorithm, we identified several Map partners, several of which were involved in the electron transport chain (ETC). Collectively, this thesis provides the first survey of the host and bacterial transcriptomes during EPEC infection, the first confirmation of the importance of the C. rodentium T2SS for robust infection in vivo, and identification of novel host mitochondrial proteins interacting with EPEC effector Map.

Text

2023-09-22

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/85999

Microbiology and Immunology

University of British Columbia

UBCV

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