Cell Surface Protein Anchoring in Gram-Positive Bacteria

描述(由申请人提供)：这项研究的长期目标是了解病原菌在感染过程中如何显示和利用表面附着蛋白，并利用这一知识开发一种治疗上有用的抗感染剂。表面蛋白通常是毒力所必需的，因为它们促进细菌黏附、抵抗吞噬细胞杀伤、宿主细胞入侵和营养获取。在革兰氏阳性细菌中，表面蛋白通过半胱氨酸转肽酶的一个大家族--排序酶以共价方式固定在细胞壁上。这项提案的研究将研究金黄色葡萄球菌中的分类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，我们将通过用分选信号类似物求解其共价复合体的核磁共振结构，研究原型索尔特酶A蛋白(SrtA)是如何识别普遍保守的LPXTG分选信号的。在目标#2中，我们将探索srtA中的活性位点变异如何控制它可以识别的排序信号。这将通过确定合理设计的单一氨基酸突变体的底物特异性来实现。在目标3中，我们将试图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个srtA小分子抑制剂进行构效关系分析，来开发这一重要酶类的抑制剂。在目标#4中，我们将研究附着在细胞壁上的索酸酶蛋白如何在感染过程中从人类血红蛋白中获得血红素铁。铁是细菌生长所必需的营养物质，在分子水平上了解血红素的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都是由远亲的纯净(靠近铁转运蛋白)结构域执行的，这些结构域进化出了不同的功能。我们的研究将研究血红素铁捕获的第一步，即血红蛋白和血红素分别与ISDH和ISDC蛋白中的整齐结构域的结合。特别是，我们将解决与亚铁血红素结合的整齐结构域的第一个结构(ISDC-亚铁血红素复合体)。 细菌利用它们在表面显示的各种不同的蛋白质感染人类。这项研究将研究这些蛋白质是如何展示的，并试图开发一种可以作为抗生素来治疗细菌感染的这一过程的抑制剂。

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.