Defense Date

2020

Document Type

Thesis

Degree Name

Master of Science

Department

Microbiology & Immunology

First Advisor

Dr. Daniel Miller

Abstract

Periodontal diseases are ubiquitous diseases. They can affect people through a chronic infection that happens over a lifetime, but also as an aggressive infection that afflicts the younger population. It not only results in tooth loss and a lower quality of life, but it can also lead to fatal secondary infections like cervical cancer, heart disease, and Alzheimer’s disease. This study looks at Selenomonas sputigena, an emerging oral pathogen, that has been hypothesized to contribute to periodontal disease. It has been linked to fatal septicemia and has been found in increased abundance within the oral biofilm during peak infection. Though S. sputigena had been discovered decades ago, there is little to no knowledge about its growth factors, both in vivo and in vitro, let alone virulence factors and disease-causing ability. In addition to establishing basic growth conditions for this understudied organism, understanding potential virulence factors for Selenomonas sputigena as an oral pathogen was the focus of this study. These potential factors for this organism include the ability to sense and make swift changes to the oral environment when it comes to pH, temperature, and oxygen concentration, sense and respond to other oral bacteria along with formation of biofilms, cell wall and potassium homeostasis, adhesion, motility, and flagellar assembly. Some of these virulence factors are proposed to be dependent upon the understudied cyclic dinucleotide, cyclic-di-AMP. Cyclic-di-AMP is the proposed secondary messenger that S. sputigena utilizes. A diadenylate cyclase, SELSP_1610, a phosphodiesterase, SELSP_2051, and two TetR family transcriptional regulators, SELSP_1770 and SELSP_1836, were all proposed based off of sequence comparison to other pathogenic oral bacteria, with special emphasis placed on SELSP_1610.

Throughout this study, we established previously unknown growth conditions within the laboratory for S. sputigena. Growth rates and growth characteristics were studied both in broth and on agar plates. The determination of oxygen sensitivity was also noted along with sensitivity to different antibiotics. Next, the focus turned to the characterization of virulence factors by studying biofilm development with self, hemagglutination and hemolysis, and binding of S. sputigena to gingival epithelial cells. After these virulence factors had been assessed, the cloning process was started and extensive assay development using reverse phase chromatography was performed. This was used to better characterize cyclic-di-AMP metabolism in S. sputigena specifically to study the enzyme, SELSP_1610, and its potential activity as a diadenylate cyclase when converting two molecules of ATP into cyclic-di-AMP. While diguanylate cyclase had been successfully studied in the past using HPLC, diadenylate cyclase had not been. This new approach required development of the assay to assess diadenylate cyclase activity by extensively adjusting the variables and conditions used. Here, we optimized enzyme kinetics, buffer conditions and concentrations, as well as the activating cations. Further results are needed to support that SELSP_1610 fits as a diadenylate cyclase. Furthermore, a competitive ELISA using a known control is also being utilized to further support the hypothesis that SELSP_1610 is a diadenylate cyclase. This foundational research will be built upon in the future to further characterize Selenomonas sputigena as a periodontal pathogen.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

12-2-2020

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