The Evolution of Lactate Catabolism and Nitric Oxide Resistance in Staphylococci

描述（由申请人提供）：金黄色葡萄球菌是美国细菌感染的主要原因，每年报告约50万例感染。临床上，S。金黄色葡萄球菌最常表现为皮肤/软组织感染，但可导致更严重的情况，如心内膜炎，骨髓炎和败血症。S.金黄色葡萄球菌是一种成功的病原体，部分原因是其能够抵抗多种宿主先天免疫效应物，包括一氧化氮（NO). S.金黄色葡萄球菌NO- 耐药性使该种与其他致病性较低的葡萄球菌成员（包括S. epidermidis和S.萨皮提库斯没有 通常通过攻击各种酶的活性位点来限制细菌生长，从而限制入侵病原体的代谢能力。S.金黄色葡萄球菌NO- 抗性需要诱导抵抗NO作用的代谢状态.我们对S.金黄色葡萄球菌- 压力S.金黄色葡萄球菌NO- 抗性是乳酸：醌氧化还原酶（Lqo），其是仅在葡萄球菌属内发现的同源酶家族的创始者。Lqo允许S.金黄色葡萄球菌使用可用的L-乳酸作为碳/能源，在面对主机NO 因此是完全毒力所必需的。而S.金黄色葡萄球菌编码单个Lqo酶，其他葡萄球菌物种具有多达四个Lqo同源物。对这些同源物的系统发育分析预测，Lqo家族是从一种类似的酶（Mqo，氧化苹果酸而不是乳酸）进化而来的，这种酶在许多细菌物种中普遍存在。在目标1中，我建议通过酶的结晶和活性位点内相关氨基酸的突变来表征Lqo与其底物的相互作用。我进一步描述了实验设计，以确定这些高度同源的酶之间的底物特异性的机制。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，我将讨论如何S。aureus将Lqo并入其NO- 抵抗代谢方案。Lqo是一种呼吸酶，需要一个末端电子受体才能发挥活性。但没有 通过与呼吸末端氧化酶的细胞色素血红素相互作用阻断有氧呼吸。S.金黄色葡萄球菌解毒NO 硝酸盐，一种厌氧末端电子受体，可能使Lqo活性在NO- 压力我已经发现，无论是主要的细胞色素aa 3氧化酶或硝酸还原酶可以支持完全NO- 抵抗因此，我将确定两个电子受体的相对贡献， 没有- 生存。此外，我将测试是否硝酸还原酶，亚硝酸还原酶和亚硝酸盐输出 参与了S.金黄色葡萄球菌NO- 抵抗最终，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，我们将查明Lqo是如何被纳入S。金黄色葡萄球菌NO- 抗性代谢方案，从而有助于病原体从寄生生物属的出现。

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.