Nitrosative Stress and Oral Bacteria

Summary Nitrite and nitric oxide are widespread and robust signaling modulators that are emerging as potential new antibacterial therapeutic agents. The oral cavity has particularly high concentrations of nitrite, which can reach 1mM. Oral microorganisms have adapted to survive such high nitrosative stress exposure and, we expect, that disruption of these adaptation mechanisms will reduce growth and survival of bacteria in the oral environment. We know that Porphyromonas gingivalis, a periodontopathogen, has high tolerance of nitrosative stress. However, the complex signaling pathways setting the basis of this tolerance are yet to be determined in in this bacterium as well as in other oral bacteria. Using whole genome expression analysis we have identified hcp encoding newly re-designated S-nitrosylase as the most dramatically upregulated gene under nitrosative stress. Furthermore, we demonstrated that regulation of Hcp is dependent on an FNR- like regulator, HcpR, that employs novel hemin-dependent mechanism to sense nitrosative stress. We hypothesize that the HcpR-Hcp system is central for adaptation of P. gingivalis to nitrosative stress. Thus, we will first define the molecular mechanisms of P. gingivalis sensing nitrosative stress through determination of the structural and biochemical characteristics of HcpR. Since adaptation to nitrosative stress involves a novel enzymatic activity mediated by Hcp, we will characterize the Hcp-mediated S-nitrosylome using proteomic approaches. In addition, we will characterize the mechanism P. gingivalis Hcp employs to mediate protection against nitrosative stress. Finally, we will investigate the contribution of other putative regulatory and effector proteins in nitrosative stress defense in P. gingivalis. It is noteworthy, that we will verify the contribution of the mechanisms under host-pathogen setting. This knowledge will provide the tools to design agents that can compromise the defense mechanisms of the periodontopathogen and turn endogenous human host nitrite and nitric oxide into a weapon that inhibits growth of the bacterium and, ultimately, we can exploit it to treat periodontal disease. We predict that this work will shed light on nitrosative stress signaling mechanisms in a variety of other bacteria that carry similar nitrosative stress protection mechanisms to those in P. gingivalis.