Cell Surface Protein Anchoring in Gram-Positive Bacteria

革兰氏阳性细菌中的细胞表面蛋白锚定

基本信息

批准号：
7767708
负责人：
Robert Thompson Clubb
金额：
$ 32.12万
依托单位：
UNIVERSITY OF CALIFORNIA LOS ANGELES
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-06-01 至 2012-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7767708
关键词：
Active Sites Affinity Amino Acids Anti-Infective Agents Antibiotics Bacteria Bacterial Adhesion Bacterial Infections Binding Biochemical Biological Assay Biological Models Catalysis Cell Surface Proteins Cell Wall Cells Chemicals Complex Coupled Cysteine Cytolysis Defense Mechanisms Development Docking Engineering Enzyme Inhibitor Drugs Enzyme Inhibitors Enzymes Erythrocytes Family Fluorescence Goals Gram-Positive Bacteria Growth Heme Heme Iron Hemoglobin Human Infection Invaded Iron Knowledge Lead Libraries Maps Membrane Proteins Methemoglobin Methods Molecular Nosocomial Infections Nutrient Organ Peptide Library Peptides Peptidyltransferase Phagocytosis Play Process Proteins Reaction Research Research Personnel Resistance Role Screening procedure Serum Signal Transduction Site-Directed Mutagenesis Sorting - Cell Movement Specificity Staphylococcus aureus Structure Structure-Activity Relationship Substrate Interaction Substrate Specificity Surface System Theft Tissues United States Variant Virulence Work analog base design glycyl-glycyl-glycine high throughput screening inhibitor/antagonist insight killings member microbial mutant novel pathogen pathogenic bacteria programs receptor research study small molecule sortase three dimensional structure transpeptidation

项目摘要

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.

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过程中如何展示和利用表面附着的蛋白质，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，表面蛋白通过分选酶（半胱氨酸转肽酶的一个大家族）共价锚定到细胞壁肽聚糖上。该提案中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，我们将通过解析其与分选信号类似物的共价复合物的 NMR 结构，研究原型分选酶 A 蛋白 (SrtA) 如何识别普遍保守的 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，我们将尝试开发这种重要酶类的抑制剂，对我们通过化合物库筛选和合理设计方法鉴定的四种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取血红素铁。铁是细菌生长的必需营养素，在分子水平上了解血红素捕获可以确定抗生素开发的新目标。这个过程中的许多步骤都是由关系较远的 NEAT（NEAr 铁转运蛋白）结构域执行的，这些结构域已进化出不同的功能。我们的研究将研究血红素铁捕获的第一步，即 IsdH 和 IsdC 蛋白中的 NEAT 结构域分别与血红蛋白和血红素结合。特别是，我们将解决与血红素结合的 NEAT 域的第一个结构（IsdC-血红素复合物）。（外行描述）细菌利用其表面展示的各种不同蛋白质来感染人类。这项研究将研究这些蛋白质如何展示，并尝试开发该过程的抑制剂，可用作治疗细菌感染的抗生素。