Functions of Pneumococcal Murein Hydrolases Required for Division and Virulence

描述(申请人提供)：肽聚糖(PG；murein)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的作用。肺炎球菌PG水解酶在应激诱导的自溶和竞争过程中的自杀性已被研究了一段时间。相比之下，人们对膜相关PG水解酶知之甚少，这种水解酶在细胞分裂过程中重塑PG，是肺炎链球菌正常生长、细胞形状、连锁和毒力所必需的。分裂PG水解酶的活性必须受到仔细的调控，以协调PG的裂解与细胞分裂的阶段，从而防止对细胞的灾难性损害。这项建议的总体目标是通过确定肺炎链球菌FtsEX：PCSB和Pmp23(PvaA)PG水解酶的功能、调节相互作用和与细胞分裂整合的机制来填补这一主要知识空白。这两个PG水解酶将被用来作为模型来检验这一提议的两个中心假设：(I)参与细胞分裂的PG水解酶是自动抑制的，其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刻解除自身抑制。这一假说最适用于FtsEX：PCSB PG水解酶，并将扩展到Pmp23，Pmp23是一种重要的分裂蛋白，可能具有裂解转糖基酶的功能。(I)通过分部PG水解酶裂解PG，允许PG合成机器进入并合成新的糖链，并在PG中为代谢PG的其他酶留下信号。PG合成的阻断可以解释PCSB或Pmp23耗尽时出现的特征圆形细胞形状。将实现两个具体目标，以实现这项提案的总体目标。目的1将确定FtsEX：PCSB复合体在肺炎球菌细胞分裂过程中的功能和信号转导，组装和与其他蛋白质的相互作用，以及在PG生物合成中的作用。目的2研究肺炎球菌Pmp23的功能和生化活性，Pmp23在PG生物合成中的作用，以及Pmp23在细胞分裂过程中的定位和与其他蛋白的相互作用。这些目标将通过一个强大的综合战略来实现，该战略结合了细菌遗传学、生理学、细胞生物学、酶学和结构生物学。这一研究结果将有助于理解这两个重要的PG水解酶在肺炎链球菌中的作用和调控，并将在细菌PG的生物合成和细胞分裂领域产生广泛的影响，特别是包括许多主要病原体的扁平椭圆形卵球菌种。FtsX蛋白可能是杀菌趋化因子的靶标，这一提议的结果将为理解这种先天免疫杀伤的机制提供信息。这项提案中的工作将验证和进一步发展卵球菌的细菌遗传学和细胞生物学方法。最后，这项提议有可能为抗生素和疫苗的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.