Nitrosative stress defenses in periodontopathogen Porphyromonas gingivalis

描述(申请人提供)：亚硝酸盐和一氧化氮在人类中广泛存在，并正在成为一种潜在的新的抗菌治疗药物。口腔中的亚硝酸盐浓度特别高，可达1 mM。口腔微生物已经适应了如此高的亚硝酸盐应激暴露，这些适应机制的破坏预计将减少细菌在口腔环境中的生长和生存。牙龈卟啉单胞菌是一种牙周病原体，以其对亚硝酸盐胁迫的高度耐受性而闻名。然而，这种耐受性的分子基础还没有得到充分的研究。利用生物信息学方法和牙龈假单胞菌暴露于亚硝酸盐和一氧化氮的微阵列分析，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。主要上调的基因是hcp，它编码一种可能的羟胺还原酶。我们进一步确定了一个被命名为HcpR的调节因子，它介导了hcp的表达，是牙龈假单胞菌在亚硝酸盐和一氧化氮存在的情况下生长所必需的。我们推测HcpR在牙周炎假单胞菌的适应中起主要作用。 亚硝化性压力。为了确定HcpR在亚硝化应激保护中的作用，我们首先定义了HcpR的调节基因及其结合所需的最小DNA序列。此外，我们还将利用核磁共振和结晶学对HcpR及其与DNA的络合物的分子结构进行研究。由于我们的研究表明牙龈假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对亚硝酸盐胁迫适应中的作用，并确定其在这种适应中的生物学功能。此外，牙龈假单胞菌编码了可能参与亚硝化应激保护或氮代谢的多个玩家。我们将确定这些角色在牙周炎假单胞菌对亚硝酸盐的适应中的作用。 压力。最后，我们将研究亚硝化胁迫在宿主-病原菌相互作用中的作用。我们的研究结果有望在调控和结构水平上提供有关牙龈假单胞菌亚硝化应激稳态机制的信息。这些知识将为设计破坏牙周病原体防御机制的药物提供工具，并将内源性人类宿主亚硝酸盐和一氧化氮转化为抑制生长的武器，最终可用于治疗牙周病。我们预测，这项工作将有助于阐明亚硝化应激自稳机制在其他多种细菌中的作用，这些细菌具有与牙龈假单胞菌相似的亚硝化应激保护机制。

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.