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，每年约有50万例感染报告。临床上,S。金黄色葡萄球菌最常表现为皮肤/软组织感染，但它可导致更严重的疾病，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许多宿主先天免疫效应器，包括一氧化氮（NO）。金黄色葡萄球菌NO-抗性将该物种与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）区分开来。NO通常通过攻击各种酶的活性位点来限制细菌的生长，从而限制入侵病原体的代谢能力。金黄色葡萄球菌NO-抗性需要诱导代谢状态抵抗NO的影响。我们对金黄色葡萄球菌在面对NO-应激时的这些代谢适应感兴趣。金黄色葡萄球菌NO-抗性所必需的一种酶是乳酸醌氧化还原酶（Lqo），它是仅在葡萄球菌属中发现的同源酶家族的创始人。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/能量来源，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，而其他葡萄球菌则含有多达四种Lqo同源物。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类似酶（Mqo，氧化苹果酸盐而不是乳酸盐）进化而来的。在Aim 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。我进一步描述了旨在定义这些高度同源酶的底物特异性机制的实验。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，需要一个终端电子受体才能发挥活性。然而，NO通过与呼吸末端氧化酶的细胞色素血红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NO胁迫下的Lqo活性增强。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可以支持完全的NO抗性。因此，我将确定两个电子受体的相对贡献

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.