Cell Surface Protein Anchoring in Gram-Positive Bacteria

描述（由申请人提供）：本研究的长期目标是了解病原菌在感染期间如何展示和利用表面附着蛋白，并利用这些知识开发治疗上有用的抗感染剂。表面蛋白通常是毒力所必需的，因为它们促进细菌粘附，抵抗吞噬细胞杀伤，宿主细胞入侵和营养获取。在革兰氏阳性菌中，表面蛋白通过分选酶共价锚定到细胞壁肽聚糖，分选酶是半胱氨酸转肽酶的一个大家族。该提案中的研究将研究金黄色葡萄球菌中的分选酶和细胞壁附着蛋白，金黄色葡萄球菌是美国医院获得性感染的主要原因。在目标#1中，我们将研究原型分选酶A蛋白（SrtA）如何通过求解其与分选信号类似物的共价复合物的NMR结构来识别普遍保守的LPXTG分选信号。在目标#2中，我们将探索SrtA中的活性位点变化如何控制它可以识别的分选信号。这将通过确定合理工程化的单个氨基酸突变体的底物特异性来实现。在目标#3中，我们将尝试通过对我们通过化合物库筛选和合理设计方法鉴定的SrtA的四种小分子抑制剂进行构效关系分析来开发这种重要酶类别的抑制剂。在目标#4中，我们将研究分选酶附着的细胞壁蛋白如何在感染期间从人类血红蛋白中获得血红素铁。铁是细菌生长的必需营养素，在分子水平上理解血红素捕获可以为抗生素的开发确定新的目标。这个过程中的许多步骤是由远亲NEAT（NEAr铁转运蛋白）结构域执行的，这些结构域已经进化出不同的功能。我们的研究将研究血红素铁捕获的第一步，分别通过IsdH和IsdC蛋白中的NEAT结构域结合血红蛋白和血红素。特别是，我们将解决NEAT结构域绑定到血红素（IsdC-血红素复合物）的第一个结构。 (Lay描述）细菌利用其表面展示的各种不同蛋白质感染人类。这项研究将研究这些蛋白质是如何展示的，并试图开发出一种抑制这一过程的抑制剂，可用作治疗细菌感染的抗生素。

DESCRIPTION (provided by applicant): The long range objective of this research is to understand how pathogenic bacteria display and utilize surface attached proteins during infections, and to use this knowledge to develop a therapeutically useful anti-infective agent. Surface proteins are frequently required for virulence, as they promote bacterial adhesion, resistance to phagocytic killing, host cell invasion, and nutrient acquisition. In gram-positive bacteria, surface proteins are covalently anchored to the cell wall peptidylglycan by sortases, a large family of cysteine transpeptidases. Research in this proposal will study the sortase enzymes and cell wall attached proteins in Staphylococcus aureus, the leading cause of hospital-acquired infections in the United States. In aim #1, we will study how the prototypical Sortase A protein (SrtA) recognizes the universally conserved LPXTG sorting signal, by solving the NMR structure of its covalent complex with a sorting signal analogue. In aim #2, we will explore how active site variations in SrtA control the sorting signals that it can recognize. This will be accomplished by determining the substrate specificities of rationally engineered single amino acid mutants. In aim #3, we will attempt to develop an inhibitor of this important enzyme class, by conducting structure activity relationship analyses on four small molecule inhibitors of SrtA that we have identified through compound library screening and rational design approaches. In aim #4 we will investigate how sortase attached cell wall proteins acquire heme iron from human hemoglobin during infections. Iron is an essential nutrient for bacterial growth and an understanding of heme capture at the molecular level could identify new targets for the development of antibiotics. Many of the steps in this process are performed by distantly related NEAT (NEAr iron Transporter) domains that have evolved distinct functions. Our research will study the first steps of heme iron capture, the binding of hemoglobin and heme by the NEAT domains in the IsdH and IsdC proteins, respectively. In particular, we will solve the first structure of a NEAT domain bound to heme (the IsdC-heme complex). (Lay Description) Bacteria infect humans using a variety of different proteins that they display on their surface. This research will study how these proteins are displayed and try to develop an inhibitor of this process that can be used as an antibiotic to treat bacterial infections.