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Gregory, Liam Patrick

University of British Columbia

Clostridioides difficile is a top threat pathogen that is highly resistant to antimicrobial drugs. Its persistence and spread rely on sporulation: differentiation of the cell into a dormant, robust particle called a spore. C. difficile sporulates when the environment is inhospitable for growth, and a spore can reconstitute into a functional cell when favourable growth conditions are restored. The spores enter and lie dormant inside the human gut, but germinate and flourish when commensal, non-resistant bacteria are killed by antimicrobials, causing serious disease. The spore is surrounded by protective layers that make it resistant to sanitation techniques, heat sterilization, and the immune system. This means they can be easily transmitted amongst patients in healthcare settings, resulting in hospital-wide outbreaks, but it also means the spores can be targeted by antimicrobials that interfere with those layers. One layer, the cortex, confers heat resistance to the spore. It is similar to the cell wall, a hugely successful antimicrobial target for other bacteria, but is only present in spores, making it a promising target for specific antimicrobial interference. This dissertation is a study of SpoVD, the penicillin-binding protein (PBP) specific to sporulation and cortex biosynthesis. Bioinformatic and x-ray crystallographic analysis of SpoVD reveal that while similar to that of other PBPs, SpoVD has several unusual structural elements. Its pedestal domain features similarities to both divisome- and elongasome-specific PBPs, which provide insights into its evolutionary history. Its catalytic serine residue is deflected away from the canonical position, resulting in a conformation associated with low catalytic efficiency and high β-lactam resistance. SpoVD also features a novel zinc-binding site, near the catalytic cleft, which may be a point of regulation with influence on the accessibility of substrates to the cleft and the efficiency of catalysis. SpoVD and the spore cortex as a whole represent an underexploited point of interference with sporulation, which is specific to only a few pathogens, and the insights herein could inform intelligent drug design. Antimicrobials targeting C. difficile SpoVD could decrease the heat resistance of spores or prevent spore shedding entirely, mitigating nosocomial transmission of this deadly pathogen.

Text

2023-11-30

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/80344

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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