Cell surface polymer display in Gram-positive bacteria

项目摘要 病原菌显示聚合性毒力因子以建立和维持感染。我们会调查的 这些聚合物的产生机理和寻找小分子聚合物组装 抑制剂。将研究革兰氏阳性细菌中的两种表面聚合物：(I)菌毛、蛋白质纤维 它从细胞表面伸出，介导与宿主组织的黏附，以及(Ii)壁磷壁酸(WTA)，高度 丰富的糖共聚物，在维持细胞壁的完整性方面发挥着重要作用。Pili和 WTA聚合物是重要的毒力因子，但人们对它们的合成知之甚少。在目标#1中- 2，我们将研究白喉棒状杆菌SpaA-菌毛原型是如何通过排序酶组装的。 聚合酶。这些酶催化一种独特的转肽反应，将相邻蛋白质共价连接起来。 菌毛亚单位通过赖氨酸等肽键结合在一起，从而赋予巨大的抗张强度，从而使 细菌粘连。通过协同使用结构、生化、细胞和化学工具，我们将 了解分类酶聚合酶如何构建菌毛轴和定义基本构件的结构 所有革兰氏阳性菌毛都是由它构成的。我们还将确定Pilus的分子基础 生物发生通过一种新的切换机制来终止，在这种机制中，菌毛在Tandemly之间转移 在细胞表面排列分类。这项工作将产生广泛的影响，作为广泛的致病因素 微生物使用类似的机制来组装它们的菌毛。在目标3中，我们将研究革兰氏阳性细菌是如何 使用Tara酶生产WTA，这是一种新的糖基转移酶，催化 聚合物生物合成。Tara是一个有希望的药物靶点，因为临床上与甲氧西林耐药葡萄球菌有关 金黄色葡萄球菌(MRSA)在宿主定植方面存在缺陷，在WTA生物合成时对β-内酰胺类药物重新敏感 被打乱了。将确定Tara的脱氧核糖核酸和底物结合形式的晶体结构，以促进 合理探索其催化机理。一种基于细胞的新型细菌的高通量筛选 还将进行细胞学分析，以发现可能有用的小分子Tara抑制剂 治疗特性。结合起来，菌毛和WTA生物发生的研究将提供对 革兰氏阳性菌中聚合物组装的化学基础，并可能导致治疗的新抗生素 由耐甲氧西林金黄色葡萄球菌和其他多重耐药细菌引起的感染。

Project Summary Pathogenic bacteria display polymeric virulence factors to establish and maintain infections. We will investigate the mechanisms through which these polymers are produced and search for small molecule polymer assembly inhibitors. Two types of surface polymers in Gram-positive bacteria will be studied, (i) pili, proteinaceous fibers that project from the cell surface to mediate adhesion to host tissues, and (ii) Wall Teichoic Acids (WTAs), highly abundant glycopolymers that play a fundamental role in maintaining the integrity of the cell wall. Both pili and WTA polymers are important virulence factors, but how they are synthesized is poorly understood. In aims #1- 2, we will study how the archetypal SpaA-pilus from Corynebacterium diphtheria is assembled by sortase polymerases. These enzymes catalyze a unique transpeptidation reaction that covalently links adjacent protein pilus subunits together via a lysine isopeptide bond, thereby conferring enormous tensile strength that enables bacterial adherence. By synergistically employing structural, biochemical, cellular and chemical tools, we will learn how sortase polymerases build the pilus shaft and define the structure of the fundamental building block from which all Gram-positive pili are constructed. We will also determine the molecular basis through which pilus biogenesis is terminated via a novel handoff mechanism in which the pilus is transferred between tandemly arranged sortases on the cell surface. This work will have a broad impact, as a wide range of pathogenic microbes use a similar mechanism to assemble their pili. In aim #3, we will study how Gram-positive bacteria produce WTA using the TarA enzyme, a novel glycosyltransferase that catalyzes the first committed step in polymer biosynthesis. TarA is a promising drug target, as clinically relevant methicillin-resistant Staphylococcus aureus (MRSA) is defective in host colonization and re-sensitized to Beta-lactam drugs when WTA biosynthesis is disrupted. Crystal structures of TarA in its apo- and substrate-bound forms will be determined, facilitating the rational exploration of its catalytic mechanism. High-throughput screening using a novel, cell-based bacterial cytological profiling assay will also be performed to discover small molecule TarA inhibitors that could have useful therapeutic properties. Combined, studies of pilus and WTA biogenesis will provide fundamental insight into the chemistry underlying polymer assembly in Gram-positive bacteria and could lead to novel antibiotics to treat infections caused by MRSA and other multi-drug resistant bacteria.