Cell surface polymer display in Gram-positive bacteria

项目摘要 病原菌显示聚合毒力因子以建立和维持感染。我们将调查 这些聚合物的生产机制和寻找小分子聚合物组装 抑制剂的革兰氏阳性菌的两种表面聚合物将被研究，（i）皮利，蛋白质纤维 从细胞表面突出以介导与宿主组织的粘附，和（ii）壁磷壁酸（WTA）， 在维持细胞壁完整性方面发挥重要作用的丰富的糖聚合物。皮利和 WTA聚合物是重要的毒力因子，但它们是如何合成的知之甚少。在目标#1- 2、研究了白喉棒状杆菌SpaA菌毛原型在分选酶作用下的组装过程 聚合酶这些酶催化一种独特的转肽反应， 菌毛亚基通过赖氨酸异肽键结合在一起，从而赋予巨大的拉伸强度， 细菌粘附。通过协同使用结构，生物化学，细胞和化学工具，我们将 了解分选酶聚合酶如何构建菌毛轴并定义基本构建块的结构 所有革兰氏阳性皮利都是由其构成的。我们还将确定菌毛 生物发生通过一种新的传递机制终止，在该机制中菌毛在串联的 在细胞表面排列分选酶。这项工作将产生广泛的影响，作为一个广泛的致病性 微生物使用类似的机制来组装它们的皮利。在目标#3中，我们将研究革兰氏阳性菌如何 使用塔拉酶生产WTA，这种酶是一种新型的糖基转移酶， 聚合物生物合成塔拉是一个有前途的药物靶点，因为临床上相关的耐甲氧西林葡萄球菌 金黄色葡萄球菌（MRSA）在宿主定植中有缺陷，当WTA生物合成时， 被打乱了将确定塔拉在其载脂蛋白和底物结合形式下的晶体结构，以促进 并对其催化机理进行了合理的探讨。使用一种新的基于细胞的细菌进行高通量筛选 还将进行细胞学谱分析以发现可能具有有用的生物学活性的小分子塔拉A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基本的洞察力， 革兰氏阳性菌中聚合物组装的化学基础，并可能导致新的抗生素治疗 由MRSA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.