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致病菌在感染过程中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药物。由于表面蛋白促进细菌粘附、抵抗吞噬细胞杀伤、宿主细胞入侵和营养获取，因此其毒力通常需要表面蛋白。在革兰氏阳性细菌中，表面蛋白通过分选酶（半胱氨酸转肽酶的一个大家族）共价固定在细胞壁肽聚糖上。本提案的研究将研究金黄色葡萄球菌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，我们将研究典型的排序酶A蛋白（SrtA）如何识别普遍保守的LPXTG排序信号，通过用排序信号类似物求解其共价复合物的NMR结构。在目标#2中，我们将探讨SrtA中的活性位点变化如何控制它可以识别的分类信号。这将通过确定合理设计的单氨基酸突变体的底物特异性来完成。在目标#3中，我们将尝试开发这一重要酶类的抑制剂，通过对我们通过化合物文库筛选和合理设计方法确定的四种SrtA小分子抑制剂进行结构活性关系分析。在目标#4中，我们将研究在感染期间，附着于分选酶的细胞壁蛋白如何从人血红蛋白中获得血红素铁。铁是细菌生长必需的营养物质，在分子水平上了解血红素捕获可以为抗生素的开发确定新的靶点。这一过程中的许多步骤是由远亲的NEAT（近铁转运蛋白）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，即IsdH蛋白和IsdC蛋白中的NEAT结构域分别结合血红蛋白和血红素。特别是，我们将解决与血红素结合的NEAT结构域的第一个结构（isdc -血红素复合物）。

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.