Regulation and impact of lipid A modification in the pathogenesis of Porphyromonas gingivalis

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Porphyromonas gingivalis is a sub-gingival Gram-negative bacterium that is closely associated with periodontitis, a chronic inflammatory disorder. Found in low numbers in healthy sites, its prevalence increases during disease, indicating it is well equipped to survive hostile inflammatory conditions. Animal models have demonstrated that P. gingivalis infection causes microbial dysbiosis in sub-gingival plaque with an ensuing increase in inflammation, which are the hallmarks of periodontal disease, marking it as a keystone pathogen. P. gingivalis evades the powerful innate immune response mediated by the receptor TLR4 by modifying its lipid A structure and has the unusual ability to elicit copious amounts of outer membrane vesicles (OMVs) armed with select virulence factors such as innate host protein destroying proteases. We have identified the crucial lipid A modification genes which are responsible for lipid A structural changes that elicit TLR4 evasion, and, additionally, contribute to OMV formation. These genes are a lipid A deacylase gene encoded by PGN_1123, and PG1773 and PG1587, which encode lipid A C1- and C4'-phosphatases respectively. In Preliminary Data, we demonstrate that all three of these genes are expressed higher in vivo in human plaque samples than in vitro and also exhibit higher expression in diseased sites than in health. However, nothing is known concerning how these genes are regulated in response to differing environmental conditions. We next demonstrate that lipid A phosphatase mutants have opposing effects on vesicle formation, suggesting regulation of PG1587 and PG1773 modulates the amount of OMVs produced. Therefore, in this application the following hypothesis will be tested: “P. gingivalis modifies its lipid A structure in response to local environmental conditions and this contributes both to its ability to survive in vivo and secrete virulence factors.” This hypothesis will be tested by the following three Specific Aims: Specific Aim 1. Characterize regulation of PGN_1123, PG1587 and PG1773 lipid A modification genes: Specific Aim 2. Determine the contribution of P. gingivalis lipid A structure and associated outer membrane molecules on OMV formation. Specific Aim 3. Comprehensive analysis of lipid A phosphatase gene expression in vivo. This work will significantly advance our knowledge of P. gingivalis pathogenesis in vivo. Future studies will include characterization of factors required for vesiculation. Overall, these studies will enable the development of diagnostic and therapeutic tools to control infection of this keystone pathogen.