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Investigating the molecular mechanisms in Clostridioides difficile colonisation
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Cheng, Jeffrey (2022) Investigating the molecular mechanisms in Clostridioides difficile colonisation. PhD thesis, University of Warwick.
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Official URL: http://webcat.warwick.ac.uk/record=b3860946
Abstract
Clostridioides difficile, a spore-forming, anaerobic bacterium is a primary cause of antibiotic associated diarrhoea. With 12,503 cases of C. difficile infections were reported in the UK between 2020-2021, the primary concern has shifted towards recurrent episodes. The molecular mechanisms of C. difficile infection are still understudied. We know little about bacterial factors that mediate colonization, an important step in infection progression. This project utilises a transposon sequencing-based approach in an in vitro human gut model to identify genes and proteins associated with colonization in C. difficile infection.
166 unique genes positively associated with colonization were identified across 3-, 6-, 12- and 24-hours, while 171 genes were either redundant or negatively associated in colonization. KEGG pathway analysis revealed the importance of metabolism and membrane proteins across these timepoints. The σB operon was identified as a contributor to colonization across all four timepoints. σB is an alternative sigma factor, responsible for the transcription of stress dependent genes. σB is regulated by anti-sigma factor, RsbW, which sequesters σB. Both σB and the uncharacterised RsbW were deemed essential in C. difficile colonization.
The role of RsbW in C. difficile infection was further investigated. As σB does not regulate its own expression in C. difficile, a deleterious fitness defect was not observed. The RsbW mutant can tolerate acidic and hypoxic environments, detoxify reactive oxygen and nitrogen species better compared to the parental strain. ∆rsbW also displayed altered sporulation rates and biofilm production, whilst an increased ability to adhere to human epithelial cells within the same in vitro gut model was observed. Interestingly, the RsbW mutant was less virulent in a Galleria mellonella infection model. Transcriptomic and RT-qPCR analysis revealed a distinct upregulation of σB-associated genes and surprisingly, the sinRR' locus. We propose a σB-indirect regulation of the sinRR' locus through the dephosphorylation spo0A by spo0E in C. difficile. Our data suggests that RsbW can indirectly control other transcriptional regulators and plays a role in C. difficile stress response, persistence and infection.
Item Type: | Thesis (PhD) | ||||
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Subjects: | Q Science > QR Microbiology | ||||
Library of Congress Subject Headings (LCSH): | Clostridium difficile, Clostridium infections -- Molecular aspects., Anaerobic bacteria -- Colonization, Biofilms, Bacterial spores | ||||
Official Date: | April 2022 | ||||
Dates: |
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Institution: | University of Warwick | ||||
Theses Department: | School of Life Sciences | ||||
Thesis Type: | PhD | ||||
Publication Status: | Unpublished | ||||
Supervisor(s)/Advisor: | Unnikrishnan, Meera ; Millard, Andrew | ||||
Format of File: | |||||
Extent: | xxi, 518 pages : illustrations | ||||
Language: | eng |
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