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

Anthony J. Clarke

Advisor Role

Principal Investigator

Abstract

Antibiotic resistance in pathogenic bacteria continues to challenge clinicians and threaten the lives of infected individuals. For this reason, new classes of antibiotics or new targets for antibiotic therapy are needed to circumvent this global health crisis. In Gram-negative bacteria, the enzymes peptidoglycan O-acetyltransferase A and B (PatA and PatB), are responsible for adding acetyl groups to the C-6 hydroxyl group of N-acetyl muramic acid (MurNAc) in peptidoglycan (PG). These acetyl groups are responsible for inhibiting the activity of lysozyme from host organisms that hydrolyze the β1-4 glycosidic linkage between MurNAc and N-acetylglucosamine (GlcNAc). The acetyl groups also regulate the rate of autolysis by precluding the activity of lytic transglycosylases, that contribute to this process. PatB from several sources has been experimentally characterized, but PatA still lacks the same amount of characterization due to difficulties with its expression and purification. Here, initial attempts to express and purify Proteus mirabilis and Campylobacter jejuni PatA are presented. Efforts to characterize functional residues of CjPatA were made by utilizing in situ assays developed to compare the resistance to lysozyme and the rates of autolysis in nutrient-deprived environments of C. jejuni strains with patA mutations. Point mutations to CjpatA were engineered for the site-specific replacements of S122, R164, H275, R283, and H315 to test the significance of these proposed catalytic residues. A preliminary application of this autolysis assay, suggested S122, H275, R283, and H315 do act as functional residues because their populations did not decrease as much as their progenitor strain, an oap operon knockout with a patA and patB complementation. O-acetylation data from the respective strains could not be generated to verify these findings.

Convocation Year

2023

Convocation Season

Fall

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