The Evolution of Lactate Catabolism and Nitric Oxide Resistance in Staphylococci

描述(申请人提供)：金黄色葡萄球菌是美国细菌感染的主要原因，每年约有50万人感染。临床上，金黄色葡萄球菌最常表现为皮肤/软组织感染，但也可导致更严重的情况，如心内膜炎、骨髓炎和败血症。金黄色葡萄球菌是一种成功的病原体，部分原因是它能够抵抗包括一氧化氮(NO)在内的许多宿主先天免疫效应物。金黄色葡萄球菌不耐药使该物种有别于其他致病性较低的葡萄球菌成员，包括表皮葡萄球菌和腐生葡萄球菌。一氧化氮通常通过攻击各种酶的活性部位来限制细菌的生长，从而限制入侵病原体的代谢能力。金黄色葡萄球菌对NO的抗性需要诱导一种对NO的影响具有抵抗力的代谢状态。我们对金黄色葡萄球菌在无压力的情况下进行的这些代谢适应很感兴趣。金黄色葡萄球菌无耐药性所必需的一种酶是一种乳酸：苯醌氧化还原酶(LQO)，它是只在葡萄球菌属中发现的一系列同源酶的创始人。LQO允许金黄色葡萄球菌在面对宿主NO时利用可用的L乳酸作为碳/能源，因此是完全毒力所必需的。金黄色葡萄球菌只编码一种Lqo酶，而其他葡萄球菌物种最多有四种Lqo同源物。对这些同源物的系统发育分析预测，Lqo家族是从许多细菌物种中常见的一种类似的酶(Mqo，氧化苹果酸而不是乳酸)进化而来的。在目标1中，我建议通过酶的结晶和活性部位内相关氨基酸的突变来表征LQO与其底物的相互作用。我进一步描述了为确定这些高度同源的酶中底物专一性的机制而设计的实验。最后，我将筛选Lqo家族的小分子抑制剂，这些化合物可能具有治疗或研究应用。因此，Lqo家族可以作为基因复制/多样化的模型进行研究，允许代谢的灵活性，并可能有助于物种形成和生态位适应。在目标2中，我将阐述金黄色葡萄球菌如何将Lqo纳入其NO抗性代谢方案。LQO是一种呼吸酶，需要一个末端电子受体才能发挥活性。然而，NO通过与呼吸终末氧化酶的细胞色素相互作用来阻断有氧呼吸。金黄色葡萄球菌将NO解毒为硝酸盐，硝酸盐是一种厌氧末端电子受体，可能使Lqo在无胁迫下具有活性。我发现，无论是主要的细胞色素Aa3氧化酶还是硝酸还原酶都能支持完全的NO抗性。因此，我将确定两个电子受体对 不--生存。另外，我将测试硝酸还原酶、亚硝酸盐还原酶和亚硝酸盐输出是否 对金黄色葡萄球菌无耐药性。最终，我们将对这一重要的葡萄球菌酶家族的结构、功能和进化有更深入的了解。此外，我们还将确定LQO是如何被结合到金黄色葡萄球菌的无抗药性代谢方案中的，从而有助于从共生生物属中出现病原体。

DESCRIPTION (provided by applicant): Staphylococcus aureus is a major cause of bacterial infection in the United States, with about half a million infections reported annually. Clinically,S. aureus most often presents as skin/soft tissue infections but it can lead to more severe conditions such as endocarditis, osteomyelitis and sepsis. S. aureus is a successful pathogen due in part to its ability to resist numerous host innate immune effectors including nitric oxide (NO). S. aureus NO-resistance distinguishes this species from other less pathogenic members of the staphylococci including S. epidermidis and S. saprophyticus. NO generally limits bacterial growth by attacking the active sites of various enzymes thereby constraining the metabolic capabilities of invading pathogens. S. aureus NO- resistance entails the induction of a metabolic state that is resistant to the effects of NO. We are interested in these metabolic adaptations mounted by S. aureus in the face of NO-stress. One enzyme necessary for S. aureus NO-resistance is a lactate:quinone oxidoreductase (Lqo), which is the founder of a family of homologous enzymes only found within the genus Staphylococcus. Lqo allows S. aureus to use available L- lactate as a carbon/energy source in the face of host NO and is therefore required for full virulence. While S. aureus encodes a single Lqo enzyme, other staphylococcal species harbor up to four Lqo homologs. Phylogenetic analyses of these homologs predict that the Lqo family evolved from a similar enzyme (Mqo, oxidizing malate rather than lactate) commonly found among many bacterial species. In Aim 1, I propose to characterize the interaction of Lqo with its substrate through crystallization of the enzyme and mutation of relevant amino acids within the active site. I further describe experiments designed to define the mechanism of substrate specificity among these highly homologous enzymes. Finally, I will screen for small-molecule inhibitors of the Lqo family, compounds that may have therapeutic or research applications. Thus, the Lqo family can be studied as a model of gene duplication/diversification allowing for metabolic flexibility and potentially contributing to speciation and niche adaptation. In Aim 2, I will address how S. aureus incorporated Lqo into its NO-resistant metabolic scheme. Lqo is a respiratory enzyme and requires a terminal electron acceptor for activity. However, NO blocks aerobic respiration by interacting with cytochrome hemes of respiratory terminal oxidases. S. aureus detoxifies NO to nitrate, an anaerobic terminal electron acceptor that might enable Lqo activity under NO-stress. I have found that either the major cytochrome aa3 oxidase or the nitrate reductase can support full NO-resistance. Accordingly, I will determine the relative contributions of both electrons acceptors to NO-survival. Also, I will test whether nitrate reductase, nitrite reductase and nitrite export are involved in S. aureus NO-resistance. In the end, we will have a deeper understanding of the structure, function and evolution of this important family of staphylococcal enzymes. Additionally, we will ascertain how Lqo was incorporated into the S. aureus NO-resistant metabolic scheme thereby contributing to the emergence of a pathogen from a genus of commensal organisms.