Functions of Pneumococcal Murein Hydrolases Required for Division and Virulence

描述（由申请人提供）：肽聚糖（PG;胞壁蛋白）水解酶在肺炎链球菌（肺炎球菌）和其他人类细菌病原体的生物学和发病机制中发挥不同作用。肺炎球菌PG水解酶介导的压力诱导的自溶和fratricide在感受态期间已经研究了一段时间。相反，很少有人知道膜相关的PG水解酶，改造PG在细胞分裂过程中，需要正常的生长，细胞形状，链，和毒力的S。肺炎。必须仔细调节分裂PG水解酶的活性以协调PG切割与细胞分裂阶段，从而防止对细胞的灾难性损伤。该提案的总体目标是通过确定功能，调节相互作用和与S的FtsEX：PcsB和Pmp 23（PvaA）PG水解酶的细胞分裂整合的机制来填补这一主要知识空白。肺炎。这两个PG水解酶将被用作模型来测试本提议的两个中心假设：（i）参与细胞分裂的PG水解酶是自抑制的，并且它们的激活严格受与调节蛋白的相互作用控制 在细胞分裂的精确时间解除自身抑制。这一假设是最发达的FtsEX：PcsB PG水解酶从以前的工作，并将扩展到Pmp 23，这是一个重要的分裂蛋白，可能作为一个裂解转糖基酶的功能。(i)通过分裂PG水解酶的PG裂解允许通过PG合成机制进入和合成新的聚糖链，并且还在PG中留下用于代谢PG的其他酶的信号。PG合成中的阻断将解释在PcsB或Pmp耗尽时发生的特征性圆形细胞形状23。为实现本提案的总体目标，将实现两个具体目标。目的1研究FtsEX：PcsB复合物在肺炎球菌细胞分裂过程中的功能、信号转导、组装、与其他蛋白的相互作用以及在PG生物合成中的作用。目的二是研究肺炎球菌Pmp 23蛋白的功能、生化活性、在PG生物合成中的作用以及在细胞分裂过程中的定位和与其他蛋白的相互作用。这些目标将通过结合细菌遗传学、生理学、细胞生物学、酶学和结构生物学的强大、全面的策略来实现。本研究结果将为进一步了解这两种重要的PG水解酶在S. pneumoniae的研究进展，将对细菌PG的生物合成和细胞分裂产生广泛的影响，特别是对长椭圆形卵球菌（Ovococcus），包括许多主要的病原菌。FtsX蛋白可能是杀菌趋化因子的靶点，该提案的结果将为理解这种先天免疫杀伤机制提供信息。本研究将为卵球菌细菌遗传学和细胞生物学方法的进一步发展提供依据。最后，这一提议有可能为抗生素和疫苗开发提供新的细菌表面靶点。

DESCRIPTION (provided by applicant): Peptidoglycan (PG; murein) hydrolases play diverse roles in the biology and pathogenesis of Streptococcus pneumoniae (pneumococcus) and other human bacterial pathogens. Pneumococcal PG hydrolases that mediate stress-induced autolysis and fratricide during competence have been studied for some time. In contrast, little is known about the membrane-associated PG hydrolases that remodel PG during cell division and are required for normal growth, cell shape, chaining, and virulence of S. pneumoniae. The activities of division PG hydrolases have to be carefully regulated to coordinate PG cleavage with stage of cell division and thereby prevent catastrophic damage to cells. The overall goal of this proposal is to fill in this major knowledge gap by determining the functions, regulatory interactions, and mechanisms of integration with cell division of the FtsEX:PcsB and Pmp23 (PvaA) PG hydrolases of S. pneumoniae. These two PG hydrolases will be used as models to test the two central hypotheses of this proposal: (i) PG hydrolases involved in cell division are autoinhibited, and their activation is strictly controlled by interactions with regulatory proteins that relieve autoinhibition at precise times during cell division. This hypothesis is most developed for the FtsEX:PcsB PG hydrolase from previous work and will be extended to Pmp23, which is an important division protein that likely functions as a lytic transglycosylase. (i) PG cleavage by division PG hydrolases allows the entry and synthesis of new glycan strands by the PG synthesis machinery and also leaves signals in the PG for other enzymes that metabolize PG. A block in PG synthesis would account for a characteristic rounded cell shape that occurs upon depletion of PcsB or Pmp23. Two specific aims will be accomplished to meet the overall goal of this proposal. Aim 1 will determine the functions and signal transduction, assembly and interactions with other proteins, and roles in PG biosynthesis of the FtsEX:PcsB complex during pneumococcal cell division. Aim 2 will determine the functions and biochemical activity, roles in PG biosynthesis, and localization and interactions with other proteins of Pmp23 during pneumococcal cell division. These aims will be accomplished by a powerful, comprehensive strategy that combines bacterial genetics, physiology, cell biology, enzymology, and structural biology. Results from this proposal will make a major contribution to understanding the roles and regulation of these two important division PG hydrolases in S. pneumoniae and will have wide-spread impact on the fields of bacterial PG biosynthesis and cell division, especially of prolate-ellipsoid shaped ovococcus species, which include many major pathogens. The FtsX protein is a likely target for bactericidal chemokines, and results from this proposal will provide information for understanding the mechanism of this innate-immune killing. Work in this proposal will provide validation and further development of methods of bacterial genetics and cell biology of ovococcus bacteria. Finally, this proposal has the potential to provide new bacterial surface targets for antibiotic and vaccine development.