Master of Science
Microbiology & Immunology
Objective: To persist in the oral cavity, the perio-pathogen Porphyromonas gingivalis must have an efficient nitrosative stress response mechanism. To better understand how P. gingivalis responds to nitrite (NO2-) we must determine what genes are involved in the nitrosative stress response network.
Methods: Through genomic screening, a list of genes was generated that were hypothesized to be part of the nitrosative stress response network and believed to be involved in nitrate/nitrite metabolism or redox state of the cells. Protein coding regions were replaced with the ermF sequence by allelic exchange mutagenesis. 15 mutants were created via electroporation and verified by genome sequencing. To test the effects of nitrosative stress on mutant and wild type strains, they were exposed to NO2- and their growth was recorded over the span of 48 hours in anaerobic conditions. Mutants were examined under different physiologically relevant conditions: with Mycoplasma and supplemented BHI media and in anaerobic and microaerophilic conditions. Normal oral keratinocytes were infected with PG_0275 mutant and wild type (WT) P. gingivalis to determine if bacterial survival was impacted by the gene deletion. Microscopy was performed on fixed normal oral keratinocytes after being infected by mutant and WT P. gingivalis.
Results: 14 of the 15 mutants did not show a significant difference in growth compared to the wild type in varying levels of NO2- in the nitrosative stress tests. PG_0275, coding for thioredoxin, showed a significant decrease of growth in NO2- compared to the control and was selected for further characterization. NO2- significantly reduced the growth of the PG_0275 mutant strain in microaerophilic (6% oxygen) conditions in Mycoplasma and sBHI media. In normal oral keratinocytes infections, the mutant bacteria had reduced survival with host cells when compared to wild type P. gingivalis. The preliminary microscopy results showed that invasion was roughly 20-30% of total bacteria for both the mutated and the wild type strains.
Conclusion: The thioredoxin, encoded by gene PG_0275, plays an important role in the nitrosative stress response of P. gingivalis. As seen in the results of the nitrosative stress tests and the infection of the normal oral keratinocytes we show that P. gingivalis gene PG_0275 is vital for survival after exposure to NO2- and within host cells. It is possible that the cysteine rich loops on the Hcp protein are being reduced by the thioredoxin donating protons. The thioredoxin protein would be working tangentially with Hcp to repair damage done by nitrosative stress. PG_0275 could also be involved in the oxidative stress response mechanism. PG_0275 is required for the bacteria to survive within a host cell. Due to NO produced in the cells P. gingivalis may use similar mechanisms to survive within cells as it uses to survive NO2- in the oral cavity. The decreased survival of mutant bacteria in host cells is due to a cell response mechanism, not because of a bacterial invasion/interaction deficiency.
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Available for download on Wednesday, May 08, 2024
Bacteriology Commons, Oral Biology and Oral Pathology Commons, Pathogenic Microbiology Commons