Defense Date
2024
Document Type
Thesis
Degree Name
Master of Science
Department
Dentistry
First Advisor
Dr. Janina P. Lewis
Second Advisor
Dr. Todd Kitten
Third Advisor
Dr. Kimberly Jefferson
Abstract
Porphyromonas gingivalis is recognized as a major etiologic agent and keystone pathogen responsible for the initiation and progression of periodontal disease. One of the distinct characteristics of oral disease is oral halitosis, representing the sulfur compounds produced by the bacteria in their pathogenic state. The overall scope of the project is to investigate the molecular mechanisms of sulfide and sulfite production in P. gingivalis starting with two selected targets. The methionine gamma lyase encoded by PG_0343 and a putative sulfite exporter encoded by PG_1572 were selected as candidate genes for the investigation of sulfur compound production. The candidate genes were knocked out of P. gingivalis strains W83 and W50, creating mutant deletion strains which were compared against their respective wildtype strains for sulfide and growth analysis.
Sulfide testing was carried out using the wildtype and methionine gamma-lyase (mgl) deficient mutants in P. gingivalis strains W83 and W50. Growth studies were conducted using the mutants and wildtype strains to explore the tolerance of the strains to increasing levels of cysteine, sulfide, and sulfite.
Proteome persulfidation in P. gingivalis was explored by harvesting protein from a wildtype culture treated with exogenous sodium hydrosulfide (NaSH) and comparing it to an untreated wildtype culture. Using a high-throughput proteomics approach, we were able to identify protein residues that are modified in the presence of NaSH treatment compared to an untreated sample, giving insight about post-translational modifications by H2S on protein thiol groups found on cysteine residues.
In the P. gingivalis W83 strain, the methionine gamma-lyase (mgl) deletion did not significantly decrease sulfide production relative to the wildtype, however, there were significant differences in sulfide production in the W50 strain, suggesting possible strain-specific variations in sulfide metabolism. The P. gingivalis W83 strain PG_1572 demonstrated inhibited growth under high levels of cysteine (5g/L). Additionally, deletion of the PG_1572 gene in the W50 strain resulted in altered growth patterns relative to the W50 wildtype. From the proteomic analysis, three proteins undergoing post-translational modifications in the presence of NaSH were identified, suggesting potential regulation of protein function under sulfide-enriched conditions.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
12-12-2024