Date on Master's Thesis/Doctoral Dissertation

8-2017

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Interdisciplinary and Graduate Studies

Degree Program

Interdisciplinary Studies (Individualized Degree), PhD

Committee Chair

Scott, David A.

Committee Member

Lamont, Richard J.

Committee Member

Potempa, Jan S.

Committee Member

Yoder-Himes, Deborah

Author's Keywords

porphyromonas gingivalis; GDM; systemic inflammation; common inherently essential genes; conditional essential genes

Abstract

This thesis contains a clinical project and distinct basic science project. Gestational diabetes mellitus occurs in 4% of pregnancies and increases the risk of birth defects, pre-term birth, and miscarriage. Gingivitis during pregnancy also increases the risk for poor pregnancy outcome. Gingivitis is a bacterial-induced disease, and specific plaque pathogens have been associated with systemic sequelae to periodontal inflammation. Therefore, we set out to monitor oral infection with three key periodontopathogens (Porphyromonas gingivalis, Filifactor alocis, and Treponema denticola) and the systemic inflammatory burden [C-reactive protein (CRP) in pregnant women with and without gingivitis and gestational diabetes. Gingivitis during pregnancy leads to a dramatic increase in systemic CRP (mean 8116 vs. 2495 ng/ml, p < 0.01), as determined by Enzyme Immunoassay (EIA). As expected, gingivitis during pregnancy was associated with oral infection with P. gingivalis, F. alocis and T. denticola and combinations thereof (all p < 0.01), as determined by salivary PCR. Gestational diabetes mellitus was also associated with increased infection with individual and multiple periodontopathogens, including P. gingivalis. Thus, diabetes and gingivitis act in concert to increase risk biomarkers for poor pregnancy outcome. Actual pregnancy outcomes in the study population are currently being monitored. The majority of cases of chronic periodontitis in developed nations are tobacco-related. For periodontal pathogens, such as P. gingivalis, to induce or exacerbate periodontal diseases in smokers, they must first be able to survive the highly complex composite toxic insult represented by cigarette smoke. While it is clear that P. gingivalis is resistant to high doses of cigarette smoke and tobacco constituents, the survival mechanisms are entirely unknown. Therefore, we first generated a P. gingivalis ATCC 33277 transposon sequencing (Tn-Seq, Library 1) and determined the putative minimal essential genome for in vitro growth in complex media in conjunction with a separate P. gingivalis ATCC 33277 Tn-Seq library generated by Klein et al. (Library 2). In all, 281 genes (61%) identified by Library 1 were common to Library 2. Many of these common genes are involved in crucial metabolic pathways, notably pyrimidine cycling as well as lipopolysaccharide, peptidoglycan, pantothenate and coenzyme A biosynthesis, and nicotinate and nicotinamide metabolism. Also in common are genes encoding heat-shock protein homologs, sigma factors, enzymes with proteolytic activity, and the majority of Sec-related protein export genes. In addition to facilitating a better understanding of critical physiological processes, transposon-sequencing technology has the potential to identify novel strategies for the control of P. gingivalis infections. Those genes defined as essential by two independently generated TnSeq mutant libraries are likely to represent particularly attractive therapeutic targets. Next, we tested Library 1 for genes essential for the survival of exposure to cigarette smoke extract. Of the 2155 P. gingivalis ATTCC 33277 genes, 257 (8%) were found to be essential for growth upon cigarette smoke exposure. The essential genes were distributed throughout the bacterial genome. Genes with products that function in DNA metabolism, energy metabolism, signal transduction, protein fate and transport and binding proteins were best represented within the essential gene set. In a competition assay of the mutants of genes of biological interest, the following mutants were outcompeted by the wild type, P. gingivalis ATCC 33277 strain: PGN_1524 encoding uncharacterized protein, PGN_1474 (luxS) encoding S-ribosyl homocysteine lyase, PGN_1200 encoding putative ATPase,PGN_1444 (carA) encoding carbamoyl- phosphate synthase small chain, PGN_0770 (rnz) encoding ribonuclease Z, PGN_0714 encoding probable pyrazinamidase/ nicotinamidase, PGN_0491 encoding probable phosphotyrosine protein phosphatase , PGN_0287 (mfa1) encoding minor fimbrium subunit Mfa1 , PGN_0088 encoding putative transcriptional regulator, and PGN_0388 (tpx) encoding putative thiol peroxidase. We confirmed the respective gene fitness in a Gifu anaerobic media conditioned with cigarette smoke extract (GAM-CSE) model. Further competition assays of the top 10 genes in the identified gene list, according to the fold change and complementation assays, could provide some of the first insights into how cigarette smoke changes the P. gingivalis phenotype in a manner likely to promote colonization and infection. Those genes identified as conditionally essential are likely to represent attractive therapeutic targets to combat P. gingivalis infection in smokers.

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