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（由申请方提供）：趋化因子是小的趋化性细胞因子，在免疫系统发育期间以及先天性和适应性免疫应答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的抗微生物肽（AMP）共享结构特征，并且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应答中起作用。然而，对于趋化因子杀死细菌的基本机制或对趋化因子杀死产生抗性的机制，人们知之甚少。本申请的目的是通过阐明趋化因子CXCL 10杀死肺炎链球菌和肺炎链球菌耐药性的分子机制来填补这些主要知识空白，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学细菌模型。本申请基于 大量未发表的数据表明，S。肺炎克雷伯氏菌对CXCL10和相关趋化因子的杀伤敏感。S的抗性。肺炎球菌对CXCL10杀伤的作用是通过跨膜FtsX分裂蛋白的细胞外环结构域中的氨基酸变化赋予的。FtsX与细胞质ATP酶FtsE和细胞外肽聚糖（PG）水解酶PcsB形成复合物，并且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。大FtsX环域（ECL 1）的NMR溶液结构已接近完成，将允许直接测试这些机制。总之，这些数据支持了本申请的中心假设，即CXCL 10与FtsX的结合异常激活PcsB PG水解酶，从而切割细胞壁并杀死细胞。这一中心假设和替代假设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制信息CXCL10耐药突变类型，并确定CXCL10杀伤模式和S.在培养物中和生物膜形成的宿主相关模型中观察到肺炎细胞。目的2将使用NMR方法和生物化学测定来确定FtsX环的结构及其与CXCL 10的相互作用以及CXCL 10是否刺激PcsB水解活性。目标3将使用显微镜方法来检查CXCL10相对于FtsEX：PcsB在肺炎球菌细胞上的结合位置，并且还将确定CXCL10中结合和杀死肺炎球菌细胞所需的氨基酸和区域。该应用的结果将首次详细研究趋化因子杀伤S. pneumoniae的CXCL10抗性的结构基础和通过FtsX的环结构域中的氨基酸变化的CXCL10抗性的结构基础。除了填补有关杀菌趋化因子与AMP相比的知识空白外，这项工作还将揭示FtsEX：PcsB PG水解酶在细胞分裂中的功能和调节，并可能为一类新的抗生素提供原型。