Mechanisms of Chemokine Killing and Resistance of Streptococcus pneumoniae

肺炎链球菌的趋化因子杀伤及耐药机制

• 批准号: 8861641
• 负责人: MALCOLM E. WINKLER
• 金额: $ 28.8万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-10 至 2019-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8861641
• 关键词: ATP phosphohydrolase; Acute; Amino Acids; Antibiotics; Bacillus anthracis; Bacteria; Bacterial Model; Binding; Biochemical; Biological; Biological Assay; CXCL10 gene; CXCL11 gene; CXCL9 gene; Cell Wall; Cell division; Cells; Charge; Cleaved cell; Collaborations; Complex; Data; Defensins; Development; Encapsulated; Event; Exhibits; Fluorescence; Fluorescence Microscopy; Genetic; Goals; Human; Hydrolysis; Immune; Immune response; Immune system; In Vitro; Infection; Inflammatory; Integral Membrane Protein; Knowledge; Laboratories; Light; Location; Membrane; Methods; Microbial Biofilms; Microscopic; Modeling; Molecular; Mutation; N-Acetylmuramoyl-L-alanine Amidase; Peptide Hydrolases; Peptidoglycan; Physiological; Play; Positioning Attribute; Proteins; Reaction; Regulation; Relative (related person); Resistance; Resistance development; Resolution; Role; Signal Transduction; Solutions; Streptococcus pneumoniae; Structure; Surface; Systems Development; Techniques; Testing; Time; Tissues; Transmembrane Domain; Variant; Work; adaptive immunity; antimicrobial peptide; bacterial resistance; bactericide; base; capsule; cell killing; cell motility; cell type; chemokine; extracellular; genetic approach; insight; killings; migration; mutant; pathogen; pathogenic bacteria; prototype; public health relevance; resistance mechanism; time use

 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.

 描述（由申请人提供）：趋化因子是小的趋化细胞因子，在免疫系统发育以及先天性和适应性免疫反应中控制免疫细胞的迁移和定位。值得注意的是，趋化因子与防御素类抗菌肽（AMP）具有相同的结构特征。 ），并且许多类型的趋化因子在体外表现出与 AMP 相当的杀菌活性，也有证据表明趋化因子的杀菌活性在其中发挥作用。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性的机制知之甚少。本申请的目的是通过阐明趋化因子杀死细菌的分子机制来填补这些主要的知识空白。趋化因子 CXCL10 杀死肺炎链球菌并对其产生耐药性，肺炎链球菌是一种主要的人类病原体，也是一种高度易处理的遗传和细胞生物细菌模型。 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作用敏感。肺炎链球菌对 CXCL10 杀伤的抵抗力是通过跨膜 FtsX 分裂蛋白的胞外环结构域的氨基酸变化而产生的。与细胞质 ATP 酶 FtsE 和细胞外肽聚糖 (PG) 水解酶 PcsB 形成的复合物，以及FtsEX:PcsB 复合物在细胞分裂中充当受调节的 PG 水解酶 FtsX 环结构域中氨基酸变化的位置与 CXCL10 结合减少或 PcsB 活化受损作为 CXCL10 抗性机制一致。环结构域 (ECL1) 已接近完成，将允许直接测试这些机制，这些数据共同支持了该应用的中心假设，即 CXCL10 结合。 FtsX 异常激活 PcsB PG 水解酶，从而裂解细胞壁并杀死细胞。这一中心假设和替代假设将通过三个特定目标进行测试，目标 1 将识别和表征新类别的机械信息 CXCL10 抗性突变，并确定 CXCL10 的模式。 Aim 2 将使用 NMR 方法和生化测定来确定培养物和宿主相关生物膜形成模型中肺炎链球菌细胞的杀灭和敏感性。确定 FtsX 环的结构及其与 CXCL10 的相互作用，以及 CXCL10 是否刺激 PcsB 水解活性。目标 3 将使用显微镜方法检查 CXCL10 相对于肺炎球菌细胞上的 FtsEX:PcsB 的结合位置，还将确定 CXCL10 中所需的氨基酸和区域。该应用的结果将提供对肺炎球菌细胞的首次详细研究。趋化因子杀死肺炎链球菌的生化机制以及 FtsX 环域氨基酸变化导致 CXCL10 耐药的结构基础 除了填补有关杀菌趋化因子与 AMP 相比的主要空白之外，这项工作还将阐明其功能和调节。 FtsEX:PcsB PG 水解酶在细胞分裂中的作用，并可能为新型抗生素提供原型。