Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Biology

Program Name/Specialization

Integrative Biology

Faculty/School

Faculty of Science

First Advisor

Dr. Joel Weadge

Advisor Role

Principle Investigator

Abstract

Bacteria inhabit many of the harshest environments on Earth; persisting and thriving in conditions thought to be unsuitable for life. One common strategy to withstand these environments is the formation of a biofilm. Biofilm composition varies greatly, depending on the underlying community that produces it. Cellulose, a polymer consistently prevalent in biofilms, has been identified as a virulence factor in many pathogens and is suspected to be involved in pathogenesis by Clostridioides difficile. C. difficile is the #1 cause of hospital acquired diarrhea, which can range from mild to life-threatening infections. Biofilm formation is hypothesized to be involved in C. difficile pathogenesis, but little is known about the process their formation. This project’s goal was to provide a basis for the understanding of C. difficile biofilms through characterization of the glycosyl hydrolase CcsZ; a protein involved Clostridial cellulose synthase complex. The first objective was to functionally characterize CcsZ, which elucidated a strong preference towards cellulosic polymers, strict endoglucanase activity, lack of thermostability and explored the role of active site residues. Objective two, the structural characterization, yielded CcsZ catalytic mutant crystals that are ready for substrate soaking and X-ray diffraction. The final objective was the live-cell analysis of CcsZ, where we provided a proof-of-concept biofilm degradation assay and began mutagenesis of the ccsZ gene in C. difficile. This thesis has characterized CcsZ, providing a basis for the understanding of biofilm synthesis in C. difficile and allowing these novel findings to be expanded on to develop new techniques to combat biofilm-mediated infections.

Convocation Year

2023

Convocation Season

Spring

Available for download on Wednesday, October 25, 2023

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