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（由适用提供）：趋化因子是小型趋化细胞因子，可控制免疫细胞系统发育过程中免疫细胞细胞的迁移和定位，以及在先天和适应性免疫电池反应中。值得注意的是，趋化因子与防御素级抗菌胡椒（AMP）共享结构特征，许多类型的趋化因子表现出与AMPS在体外相当的细菌活性。也有证据表明趋化因子杀菌活性在先天免疫反应中起着作用。然而，关于趋化因子杀死细菌或抗趋化因子杀死的机制的基本机制知之甚少。该应用的目的是填补这些主要空白，以通过阐明趋化因子CXCL10杀伤和肺炎链球菌的耐药性的分子机制来填补这些主要差距，该机制也是一种主要的人类病原体，它也是一种高度狭窄的遗传和细胞生物学细菌模型。此应用程序是基于的 在大量未发表的数据上，表明肺炎链球菌对CXCL10和相关趋化因子杀死敏感。肺炎链球菌对CXCL10杀伤的抗性是由跨膜FTSX分裂蛋白的细胞外环域的氨基酸变化所赋予的。 FTSX与细胞质ATPase FTSE和细胞外肽聚糖（PG）水解酶PCSB以及FTSEX：PCSB复合物在细胞分裂中充当调节的PG水解酶。 FTSX回路结构域中氨基酸变化的位置与改善的CXCL10结合或PCSB激活受损作为CXCL10电阻的机制一致。大型FTSX循环结构域（ECL1）的NMR溶液结构接近完成，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与FTSX的结合异常激活PCSB PG水解酶，从而裂解细胞壁和杀死细胞。这一中心假设和替代假设将通过三个特定目的来检验。 AIM 1将识别并表征新的机械信息丰富的CXCL10抗性突变，并确定培养物中肺炎链球菌细胞的CXCL10杀伤和敏感性的模式以及生物膜形成的宿主相关模型。 AIM 2将使用NMR方法和生化测定来确定FTSX环的结构及其与CXCL10的相互作用，以及CXCL10是否刺激PCSB水解活性。 AIM 3将使用微观方法检查CXCL10相对于肺炎球菌细胞上的FTSEX：PCSB结合的位置，还将确定肺炎细胞结合和杀死肺炎细胞所需的CXCL10中的氨基酸和区域。该应用的结果将对肺炎链球菌杀死的生理和生化机制提供首次详细研究，以及氨基酸在FTSX环路域中氨基酸变化CXCL10抗性的结构基础。除了填补与AMP相比的细菌趋化因子知识的主要空白外，这项工作还阐明了FTSEX：PCSB PG水解酶在细胞分裂中的功能和调节，并可能为新的抗生素提供原型。