Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Chemistry

Program Name/Specialization

Biological and Chemical Sciences

Faculty/School

Faculty of Science

First Advisor

Dr. Anthony J. Clarke

Advisor Role

Principal Investigator

Abstract

The bacterial cell wall is a highly organized structure essential for maintaining cell shape, resisting osmotic stress, and preserving bacterial viability. Its main component, peptidoglycan (PG), has long been an effective antibiotic target. However, the rise of antimicrobial resistance has increased the need for alternative therapeutic strategies. One promising approach is to target virulence-associated pathways rather than bacterial viability directly. O-Acetylation of PG protects the bacterial cell wall from host lysozyme and limits cleavage by endogenous lytic transglycosylases (LTs), thereby contributing to bacterial persistence and pathogenicity. In the Gram-negative pathogen Campylobacter jejuni, this modification is mediated by PatA and PatB, but the relationship between PatA structure and function remains poorly understood.

Previous studies showed that wild-type C. jejuni contains more PG O-acetylation than a patA deletion mutant (ΔpatA), confirming the importance of PatA for modification of the PG sacculus. However, functional characterization of PatA in vitro has been limited by the difficulty of purifying this integral membrane protein. To address this limitation, an in situ complementation-based assay was developed to measure the ability of CjPatA variants to protect cells from EDTA-induced autolysis. EDTA destabilized the outer membrane and promoted the release of endogenous LTs, increasing degradation of susceptible PG. Compared with wild-type and complemented strains, ΔpatA exhibited more rapid turbidity loss and, by colony-forming unit enumeration, a greater loss of recoverable cells.

The assay was validated using ΔpatA strains complemented with patA variants carrying substitutions of the proposed catalytic residues His315 and Tyr455; both variants produced phenotypes closely resembling the autolysis-susceptible ΔpatA strain. When additional tunnel-associated substitutions were introduced into the same ΔpatA complementation background, W274A, R283A, and N293A caused the strongest defects, supporting the functional importance of residues positioned within the predicted intramembrane tunnel. These functional effects were consistent with structural modelling and docking analyses, which implicated Arg283, Asn293, and His336 in possible ligand interactions or positioning within the tunnel. Ser122, His336, His275, and Ser311 made substantial but nonessential contributions to CjPatA function, whereas substitution of Arg164, Lys268, and Ser279 was comparatively well tolerated. Direct PG analysis confirmed that the H315A and Y455F complemented variants had O-acetylation levels similar to ΔpatA, supporting the conclusion that their autolysis phenotypes resulted from impaired CjPatA-dependent PG O-acetylation. Together, these findings support the functional importance of the predicted CjPatA tunnel and provide a foundation for further investigating PG O-acetylation as an antivirulence target.

Convocation Year

2027

Convocation Season

Spring

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Available for download on Wednesday, November 03, 2027

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