Nitrosative Stress and Oral Bacteria

摘要 亚硝酸盐和一氧化氮是广泛和强大的信号调节器，正在成为 潜在的新型抗菌治疗药物。口腔中的浓度特别高 亚硝酸盐，可达1 mm。口腔微生物已经适应了在如此高的温度下生存 亚硝化应激暴露，我们预计，这些适应机制的破坏将 减少口腔环境中细菌的生长和存活。我们知道卟啉单胞菌 牙周炎是一种牙周病原体，对亚硝酸盐胁迫有很高的耐受性。然而，这个综合体 在这种细菌中，建立这种耐受性基础的信号通路尚未确定。 在其他口腔细菌中也是如此。通过全基因组表达分析，我们鉴定出了hcp 编码新被重新指定的S-亚硝酸酯酶基因 亚硝化性压力。此外，我们还证明了Hcp的调节依赖于FNR- 与调节因子HcpR一样，HcpR使用新的氯化血红素依赖机制来感知亚硝化应激。 我们推测HcpR-Hcp系统是牙龈假单胞菌适应亚硝酸盐的中心。 压力。因此，我们将首先确定牙龈假单胞菌感知亚硝酸盐的分子机制。 通过测定HcpR的结构和生化特性来进行应激。自.以来 对亚硝化压力的适应涉及一种由Hcp介导的新的酶活性，我们将 用蛋白质组学方法研究Hcp介导的S-亚硝基组。此外，我们还将 牙龈假单胞菌Hcp介导亚硝酸盐保护作用的机制 压力。最后，我们将调查其他假定的监管和效应者的贡献。 牙龈假单胞菌亚硝化应激防御中的蛋白质。值得注意的是，我们将核实 寄主-病原菌环境下的作用机制。这些知识将提供工具 设计能够破坏牙周病原体防御机制的试剂，并 将内源性人类宿主亚硝酸盐和一氧化氮转化为抑制肿瘤生长的武器 细菌，最终，我们可以利用它来治疗牙周病。我们预测这项工作 将阐明携带亚硝化应激信号的各种其他细菌的机制 亚硝化应激保护机制与牙龈假单胞菌相似。

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.