Mechanisms of Chemokine Killing and Resistance of Streptococcus pneumoniae

 描述(申请人提供)：趋化因子是一种小的趋化性细胞因子，在免疫系统发育过程中控制免疫细胞的迁移和定位，并在先天性和获得性免疫反应中控制。值得注意的是，趋化因子具有防御素类抗菌肽(AMPs)的结构特征，许多类型的趋化因子在体外显示出与AMP相当的杀菌活性。也有证据表明，趋化因子杀菌活性在先天免疫反应中发挥作用。然而，人们对趋化因子杀死细菌的基本机制或对趋化因子杀伤产生抗药性的机制知之甚少。这项应用的目标是通过阐明趋化因子CXCL10杀死肺炎链球菌和耐药的分子机制来填补这些主要的知识空白。肺炎链球菌是一种主要的人类病原体，也是一个高度易处理的遗传和细胞生物学细菌模型。此应用程序基于 大量未发表的数据表明肺炎链球菌对CXCL10和相关趋化因子的杀伤敏感。肺炎链球菌对CXCL10杀伤的抵抗力是通过跨膜FtsX分裂蛋白胞外环区的氨基酸变化来实现的。FtsX与胞内ATPase FTSE和胞外肽聚糖(PG)水解酶PCSB形成复合体，FtsEX：PCSB复合体在细胞分裂过程中发挥调节PG水解酶的作用。FtsX环区氨基酸变化的位置与CXCL10结合减少或PCSB激活受损是CXCL10抗性的机制一致。大的FtsX环结构域(ECL1)的核磁共振溶液结构接近完成，将允许直接测试这些机制。综上所述，这些数据支持这一应用的中心假设，即CXCL10与FtsX结合后异常激活PCSB PG水解酶，从而裂解细胞壁并杀死细胞。这一中心假说和备选假说将通过三个具体目标进行检验。目的1将鉴定和表征新的机械信息性CXCL10耐药突变类型，并确定CXCL10在培养和与宿主相关的生物被膜形成模型中的杀灭模式和肺炎链球菌细胞的敏感性。目的2将使用核磁共振方法和生化分析来确定FtsX环的结构及其与CXCL10的相互作用，以及CXCL10是否刺激PCSB的水解活性。AIM 3将使用显微镜方法检查CXCL10与肺炎球菌细胞上FtsEX：PCSB的结合位置，并将确定CXCL10中结合和杀死肺炎球菌细胞所需的氨基酸和区域。这一应用的结果将首次详细研究趋化因子杀死肺炎链球菌的生理生化机制，以及通过FtsX环区氨基酸的变化来对抗CXCL10的结构基础。除了填补有关杀菌趋化因子与AMP相比的主要知识空白外，本工作还将阐明FtsEX：PCSB PG水解酶在细胞分裂中的功能和调控，并可能为一类新的抗生素提供原型。

 DESCRIPTION (provided by applicant): Chemokines are small chemotactic cytokines that control the migration and positioning of immune cells during immune system development and in the innate and adaptive immune responses. Notably, chemokines share structural features with the defensin-class of antimicrobial peptides (AMPs), and many types of chemokines show bactericidal activity comparable to AMPs in vitro. There is also evidence suggesting that chemokine bactericidal activity plays roles in innate immune responses. Yet, relatively little is known about the basic mechanisms by which chemokines kill bacteria or the mechanisms by which resistance develops to chemokine killing. The goal of this application is to fill in these major gaps in knowledge by elucidating the molecular mechanisms of chemokine CXCL10 killing and resistance of Streptococcus pneumoniae, which is a major human pathogen that also serves as a highly tractable genetic and cell biological bacterial model. This application is based on a large body of unpublished data showing that S. pneumoniae is sensitive to killing by CXCL10 and related chemokines. Resistance of S. pneumoniae to CXCL10 killing is imparted by amino acid changes in extracellular loop domains of the transmembrane FtsX division protein. FtsX forms a complex with cytoplasmic ATPase FtsE and extracellular peptidoglycan (PG) hydrolase PcsB, and the FtsEX:PcsB complex functions as a regulated PG hydrolase in cell division. The locations of the amino acid changes in the FtsX loop domains are consistent with decreased CXCL10 binding or impaired PcsB activation as mechanisms of CXCL10 resistance. An NMR solution structure of the large FtsX loop domain (ECL1) is near completion and will allow direct testing of these mechanisms. Together, these data support the central hypothesis of this application that CXCL10 binding to FtsX aberrantly activates PcsB PG hydrolase, thereby cleaving cell walls and killing cells. This central hypothesis and alternate hypotheses will be tested by three specific aims. Aim 1 will identify and characterize new classes of mechanistically informative CXCL10-resistant mutations and determine the mode of CXCL10 killing and sensitivity of S. pneumoniae cells in culture and in a host-relevant model of biofilm formation. Aim 2 will use NMR methods and biochemical assays to determine the structure of the FtsX loops and their interactions with CXCL10 and whether CXCL10 stimulates PcsB hydrolysis activity. Aim 3 will use microscopic methods to examine where CXCL10 binds relative to FtsEX:PcsB on pneumococcal cells and will also determine amino acids and regions in CXCL10 required for binding and killing of pneumococcal cells. Results from this application will provide the first detailed study of the physiological and biochemical mechanisms of chemokine killing of S. pneumoniae and the structural basis for CXCL10 resistance by amino acid changes in loop domains of FtsX. Besides filling in major gaps in knowledge about bactericidal chemokines compared to AMPs, this work will shed light on the function and regulation of the FtsEX:PcsB PG hydrolase in cell division and possibly provide a prototype for a new class of antibiotics.