Supramolecular Complexes That Mediate Pneumococcal PG Biosynthesis and Virulence

描述（由申请人提供）：肺炎链球菌（肺炎球菌）是一种极其严重的人类呼吸道病原体，每年在全球造成200多万人死亡。肺炎链球菌临床分离株的多药耐药性正在以惊人的速度增加。许多临床相关的抗生素，包括内酰胺类和万古霉素，都是针对肽聚糖（PG）的生物合成。PG在细胞壁中形成主要的刚性结构，决定细胞的形状和大小，并作为其他肺炎球菌毒力因子（包括荚膜、磷壁酸和排序酶转移蛋白）共价附着的支架。尽管其在生理学和发病机制上具有重要意义，但人们对介导肺炎链球菌和其他椭球状革兰氏卵球菌阳性病原体细胞表面PG生物合成的超分子蛋白复合物知之甚少。该项目的长期目标是填补关于介导肺炎球菌PG生物合成的超分子蛋白复合物的位置、相互作用、调节动力学和功能的主要知识空白。这一建议是基于大量的新论文和未发表的数据，这些数据证明了肺炎链球菌中PG生物合成的许多独特和意想不到的特性。PG生物合成是一个广泛的课题，包括PG合成和PG水解重塑。这项为期五年的计划包括四个协同的特定目标，以解决卵球菌PG生物合成中一些最重要的突出问题。这四个目标在概念上与PG合成和PG重塑酶作为动态超分子复合物起作用的中心假设有关，其活动和相互作用相互编排并与细胞分裂有关。之所以选择这些目标，是因为它们将产生关于PG生物合成的基本原理，得到强有力的新数据的支持，在实验上易于处理，相互促进，并且将对该领域产生重大影响。相关目的1和2将阐明青霉素结合蛋白（PBPs）如何通过与少量必需主组织蛋白的相互作用定位、激活并与细胞分裂阶段相结合。目的3将验证参与PG重塑的PG水解与细胞分裂直接耦合的假设。目的4将确定PG水解酶如何调节PBPs使用的PG五肽底物的供应，以及PG肽是否在组织PG合成中发挥作用。一个综合的策略，结合了创新的遗传、生化、细胞生物学和微观方法的结果，将用于满足这些目标，为这个主要的细菌病原体。该研究结果将挑战和扩展肺炎链球菌和其他卵球菌病原体中PG生物合成的范式和模型。由于细胞表面对肺炎球菌的毒力至关重要，而且胞浆外蛋白是可获得的和可药物化的，因此本研究中研究的一些关键的PG合成和重塑蛋白有望成为新的抗生素和疫苗候选蛋白。

DESCRIPTION (provided by applicant): Streptococcus pneumoniae (pneumococcus) is an extremely serious human respiratory pathogen that kills well over two million people annually worldwide. Multidrug resistance is increasing in S. pneumoniae clinical isolates at an alarming rate. Many clinically relevant antibiotics, including ¿-lactams and vancomycin, target peptidoglycan (PG) biosynthesis. PG forms the major rigid structure in the cell wall that determines cell shape and size and serves as the scaffolding onto which other pneumococcal virulence factors are covalently attached, including capsule, teichoic acids, and sortase-transferred proteins. Despite its importance to physiology and pathogenesis, little is known about the supramolecular protein complexes that mediate PG biosynthesis on the cell surface of S. pneumoniae and other ellipsoid-shaped ovococcus Gram-positive pathogens. The long-term goal of this project is to fill in this major knowledge gap about the locations, interactions, regulatory dynamics, and functions of the supramolecular protein complexes that mediate pneumococcal PG biosynthesis. This proposal is based on a large body of new papers and unpublished data that demonstrates numerous unique and unexpected properties of PG biosynthesis in S. pneumoniae. PG biosynthesis is a broad topic that encompasses both PG synthesis and PG remodeling by hydrolysis. This five- year proposal consists of four synergistic Specific Aims that address some of the most important outstanding problems in ovococcus PG biosynthesis. These four Aims are conceptually linked to the central hypothesis that PG synthesis and PG remodeling enzymes function as dynamic supramolecular complexes, whose activities and interactions are choreographed with each other and with cell division. These Aims were chosen, because they will yield fundamental principles about PG biosynthesis, are supported by strong new data, are experimentally tractable, feed into each other, and will have high impact on the field. Related Aims 1 and 2 will elucidate how penicillin binding proteins (PBPs) are localized, activated, and tied to stages of cell division through interactions with a small number of essential master organizer proteins. Aim 3 will test the hypothesis that PG hydrolysis involved in PG remodeling is coupled directly to cell division. Aim 4 will determine how PG hydrolases modulate the supply of PG pentapeptide substrates used by PBPs and whether PG peptides play roles in organizing PG synthesis. A comprehensive strategy that combines results from innovative genetic, biochemical, cell biology, and microscopic approaches with those from colonization and infection models will be used to meet these Aims for this primary bacterial pathogen. Results from this proposal will challenge and expand paradigms and models about PG biosynthesis in S. pneumoniae and other ovococcus pathogens. Since the cell surface is critical to pneumococcal virulence and extracytoplasmic proteins are accessible and druggable, there is an expectation that some of the critical PG synthesis and remodeling proteins studied in this proposal will emerge as new antibiotic and vaccine candidates.