Nitrosative stress defenses in periodontopathogen Porphyromonas gingivalis

描述（由申请人提供）：亚硝酸盐和一氧化氮在人体中广泛存在且含量丰富，正在成为一种潜在的新型抗菌治疗剂。口腔中的亚硝酸盐浓度特别高，可以达到1 mM。口腔微生物已经适应于在这种高亚硝化应激暴露下存活，并且这些适应机制的破坏将预期减少口腔环境中细菌的生长和存活。牙龈卟啉单胞菌是一种牙周病病原菌，对亚硝化应激具有高度耐受性。然而，这种耐受性的分子基础研究不足。使用生物信息学方法和暴露于亚硝酸盐和一氧化氮的牙龈卟啉单胞菌的微阵列分析，我们确定了几个可能在亚硝化应激保护中发挥作用的潜在候选者。主要的上调基因是hcp，它编码一个假定的羟胺还原酶。我们进一步鉴定了一种调节剂，命名为HcpR，其介导hcp的表达，并且是在亚硝酸盐和一氧化氮存在下牙龈卟啉单胞菌生长所必需的。我们假设HcpR是牙龈卟啉单胞菌适应性的主要参与者 亚硝化应激为了确定HcpR在亚硝化胁迫保护中的作用，我们首先定义HcpR的调节子及其结合所需的最小DNA序列。此外，我们将进行HcpR及其与DNA的复合物的分子结构研究，使用NMR和晶体学。由于我们的研究表明牙龈卟啉单胞菌的主要调控基因是hcp，我们将确定其基因产物在细菌适应亚硝化应激中的作用，并确定其在这种适应中的生物学功能。此外，牙龈卟啉单胞菌编码可能参与亚硝化应激保护或氮代谢的多个参与者。我们将描述并确定这些参与者在牙龈卟啉单胞菌适应亚硝化中的作用。 应力最后，我们将研究亚硝化应激在宿主-病原体相互作用中的作用。我们的研究结果预计将提供有关亚硝化应激稳态机制的牙龈卟啉单胞菌在监管和结构水平的信息。这方面的知识将提供工具，以设计代理商，妥协的牙周病原体的防御机制，并把内源性人类宿主亚硝酸盐和一氧化氮成为武器，抑制生长，并最终可以利用治疗牙周疾病。我们预测，这项工作将揭示亚硝化应激稳态机制在各种其他细菌携带类似的亚硝化应激保护机制的牙龈卟啉单胞菌。

DESCRIPTION (provided by applicant): Nitrite and nitric oxide are widespread and abundant in humans and are emerging as a 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 disruption of these adaptation mechanisms would be expected to reduce growth and survival of bacteria in the oral environment. Porphyromonas gingivalis, a periodontopathogen, is well known for its high tolerance of nitrosative stress. However, the molecular basis of this tolerance is under-investigated. Using bioinformatics approaches and microarray analysis of P. gingivalis exposed to nitrite and nitric oxide, we identified several potential candidates that may play a role in nitrosative stress protection. The major upregulated gene was hcp, which codes for a putative hydroxylamine reductase. We have further identified a regulator, designated HcpR, which mediates expression of hcp and is required for growth of P. gingivalis in the presence of both nitrite and nitric oxide. We hypothesize that HcpR is a major player in adaptation of P. gingivalis to nitrosative stress. To determine its role in nitrosative stress protection, we will first definethe regulon of HcpR and the minimum DNA sequence required for its binding. Furthermore, we will carry out molecular structure studies of HcpR and of its complexes with DNA using NMR and crystallography. Since our study shows that the major regulated gene in P. gingivalis is hcp, we will determine the role of its gene product in adaptation of the bacterium to nitrosative stress an define its biological function in such adaptation. Also, P. gingivalis codes for multiple players that may be involved in nitrosative stress protection or nitrogen metabolism. We will characterize and determine the roles of those players in adaptation of P. gingivalis to nitrosative stress. Finally, we will investigate the role of nitrosative stress in host-pathogen interactions. The results of our study are expected to provide information regarding nitrosative stress homeostasis mechanisms in P. gingivalis at the regulatory and structural levels This knowledge will provide the tools to design agents that compromise the defense mechanisms of the periodontopathogen and turn endogenous human host nitrite and nitric oxide into a weapon that inhibits growth and ultimately can be exploited to treat periodontal disease. We predict that this work will shed light on nitrosative stress homeostasis mechanisms in a variety of other bacteria that carry similar nitrosative stress protection mechanisms to those in P. gingivalis.