Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Chemistry

Faculty/School

Faculty of Science

First Advisor

Dr. Michael Suits

Advisor Role

Supervisor

Abstract

Dental biofilms are coupled polymicrobial aggregates that have attached to solid surfaces in the oral cavity. These collections of microorganisms are known to cause periodontal diseases that commence as localized inflammation of the gingiva and if untreated, eventually lead to irreversible alveolar bone resorption and tooth loss. Porphyromonas gingivalis is one of three periodontal pathogens that make up the “Red Complex”; a bacterial consortium responsible for the production of polysaccharide-rich biofilms that are essential to the inception and progression of periodontal disease. The dysbiosis and destructive inflammation caused by these organisms propel a self-sustained feed-forward loop that perpetuates periodontal disease. Antibiotic misuse and the dense polysaccharide network of biofilms have resulted in widespread antibiotic resistances, which epitomizes the need for the identification and application of new therapeutic targets in order to attenuate bacterial growth and degrade existing biofilm structures. Previously reported transcriptomics analysis was utilized to determine which P. gingivalis transcripts were upregulated during biofilm development. For the present research program, three gene products, PGN_0427, PGN_0982 and PGN_1461, were chosen for functional and structural characterization to elucidate their roles in contributing to P. gingivalis biology. Bioinformatics analyses suggested PGN_0427 to be a glycosyl hydrolase belonging to family 57, a group of proteins capable of hydrolysing the glycosidic bond between two or more carbohydrates. PGN_0982 was suggested to be a putative Mrr restriction system protein. PGN_1461 was predicted to be a contain a fused spore maturation protein domain, thought to play a role in the transport of iron (Fe2+) across the cell membrane. Codon-optimized genes were expressed in Escherichia coli BL21 cells and these recombinant proteins were purified for functional analysis as well as structural elucidation via X-ray diffraction analysis. Although no usable diffraction data are available for PGN_0427, various carbohydrate-activity assays suggested the enzyme to be specific for pullulan. High-resolution diffraction data (1.65Å) has been collected for PGN_0982, although no crystal structure has been elucidated due to the lack of a suitable homology model for molecular replacement. DNA degradation assays visualized by agarose gel electrophoresis support the notion that PGN_0982 possesses restriction endonuclease activity. PGN_1461 was a collaboration with James Stevenson for which high-resolution diffraction data (1.87Å) has been collected.

Convocation Year

2019

Convocation Season

Fall

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