Signal Transduction by Essential VicRKX in Pneumococcus

肺炎球菌中必需 VicRKX 的信号转导

• 批准号: 7748997
• 负责人: MALCOLM E. WINKLER
• 金额: $ 35.36万
• 依托单位: TRUSTEES OF INDIANA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-01-01 至 2011-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7748997
• 关键词: Affect; Anabolism; Animal Model; Animals; Antibiotic Resistance; Antibiotics; Bacteria; Binding; Biochemical; Biochemical Genetics; Biochemical Reaction; Cell Wall; Cell surface; Cells; Cytoplasm; DNA; Data; Enzymes; Flavin Mononucleotide; Fluorescence Anisotropy; Future; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Screening; Genetic Transcription; Goals; Grant; Growth; Human; In Vitro; Infection; Knowledge; LacZ Genes; Lactamase; Measures; Mediating; Membrane Proteins; Metabolic; Metals; Methods; Nature; Peptidoglycan; Phosphoric Monoester Hydrolases; Phosphorylation; Physiological; Physiology; Play; Promoter Regions; Property; Proteins; Pyruvate Oxidase; Reaction; Regulation; Regulon; Reporter; Research Personnel; Resistance development; Role; Running; Screening procedure; Signal Transduction; Signal Transduction Pathway; Site; Streptococcus; Streptococcus pneumoniae; Stress; Surface; System; Testing; Time; Transcriptional Activation; Transcriptional Regulation; Transferase; Virulence; Virulence Factors; acetyl phosphate; base; extracellular; feeding; genetic selection; in vivo; insight; member; mutant; pathogen; programs; protein-histidine kinase; research study; respiratory; response; small molecule; vaccine development; yeast two hybrid system; Affect; Anabolism; Animal Model; Animals; Antibiotic Resistance; Antibiotics; Bacteria; Binding; Biochemical; Biochemical Genetics; Biochemical Reaction; Cell Wall; Cell surface; Cells; Cytoplasm; DNA; Data; Enzymes; Flavin Mononucleotide; Fluorescence Anisotropy; Future; Gene Expression; Gene Targeting; Genes; Genetic; Genetic Screening; Genetic Transcription; Goals; Grant; Growth; Human; In Vitro; Infection; Knowledge; LacZ Genes; Lactamase; Measures; Mediating; Membrane Proteins; Metabolic; Metals; Methods; Nature; Peptidoglycan; Phosphoric Monoester Hydrolases; Phosphorylation; Physiological; Physiology; Play; Promoter Regions; Property; Proteins; Pyruvate Oxidase; Reaction; Regulation; Regulon; Reporter; Research Personnel; Resistance development; Role; Running; Screening procedure; Signal Transduction; Signal Transduction Pathway; Site; Streptococcus; Streptococcus pneumoniae; Stress; Surface; System; Testing; Time; Transcriptional Activation; Transcriptional Regulation; Transferase; Virulence; Virulence Factors; acetyl phosphate; base; extracellular; feeding; genetic selection; in vivo; insight; member; mutant; pathogen; programs; protein-histidine kinase; research study; respiratory; response; small molecule; vaccine development; yeast two hybrid system

Streptococcus pneumoniae (pneumococcus) is an important gram positive human respiratory pathogen that is developing antibiotic resistance. The essential VicRK two component system (TCS) and its associated third component VicX are required for pneumococcal virulence. The long-term goal of this proposal is to determine the signal transduction pathways used by the VicRKX system to regulate genes encoding an essential murein biosynthetic enzyme and established virulence factors on the pneumococcal cell surface. Our new results show that this regulation is direct and mediated by phosphorylation of the VicR response regulator (RR). Other new results suggest that defective cell wall biosynthesis may generate metabolic signals sensed by the VicRKX system, possibly by the VicK histidine kinase (HK), which lacks an extracellular sensing domain but contains a PAS domain, or by other phosphoryl group donors. Five Specific Aims will be achieved in this five-year grant: Aim I, We will characterize the binding of phosphorylated VicR and the resulting transcription activation at promoter regions of key regulon gene members using in vitro biochemical methods. Aim II. We will determine the expression levels of VicRKX proteins and the virulence properties of vicRKX mutants to understand why the VicK HK is not essential in S. pneumoniae growing in culture, but is required for virulence. We will use genetic approaches to determine which other donors phosphorylate the VicR RR in the absence of the VicK HK. Aim III. We will use biochemical approaches to determine the signal(s) sensed by the VicK HK and whether the VicK HK possesses a VicR-P phosphatase activity. We will construct lacZ reporter fusions to key regulon genes to determine culture and stress conditions that may be sensed by the VicRKX system. Genetic screens and selections will be used to identify possible signals sensed by the VicRKX system and other modes of regulation of these virulence factor genes. Aim IV. We will use biochemical and genetic approaches to determine the roles of the VicX third component and putative VicK phosphatase activity in VicRKX signal transduction. We will determine whether the (3-lactamase fold in VicX plays a role in sensing additional signals. Aim V. We will use biochemical and microarray methods to determine new members of the VicRKX regulon that were missed in previous studies. This grant will provide fundamental new knowledge about the regulation of important cell wall biosynthesis and virulence factor genes in a serious human pathogen. It will provide insights into the multiple mechanisms of signal transduction used by the VicRKX system in pneumococcus and likely other species of streptococcus to communicate between the cytoplasm and cell surface. Understanding the unusual features of VicRKX signal transduction will extend the paradigm of TCS regulation. Finally, gene products in the VicRKX regulon are promising surface targets for future antibiotic and vaccine development.

肺炎链球菌（肺炎球菌）是一种重要的革兰氏阳性人类呼吸道病原体 正在发展抗生素耐药性。必需的VICRK两个组件系统（TCS）及其相关 肺炎球菌毒力需要第三个成分VICX。该提议的长期目标是 确定VICRKX系统用于调节编码基因的信号转导途径 必需的murein生物合成酶和肺炎球菌表面上建立的毒力因子。 我们的新结果表明，该调节是由VICR响应的磷酸化直接和介导的 调节器（RR）。其他新结果表明，有缺陷的细胞壁生物合成可能会产生代谢 VICRKX系统感测的信号可能是Vick组氨酸激酶（HK），它缺乏 细胞外感应结构域，但包含一个PAS结构域或其他磷酸组供体。五个具体 目标将在这项五年的赠款中实现：目标I，我们将表征磷酸化VICR的结合 以及使用体外的关键调节基因构件的启动子区域的转录激活 生化方法。目标II。我们将确定VICRKX蛋白的表达水平和毒力 VICRKX突变体的特性，以了解为什么Vick HK在肺炎链球菌中并不必不可少 培养，但毒力是必需的。我们将使用遗传方法来确定哪些其他捐助者 在没有Vick HK的情况下，将VICR RR磷酸化。目标三。我们将使用生化方法 确定Vick HK感应的信号以及Vick HK是否具有VICR-P-P磷酸酶 活动。我们将构建LACZ报告基因融合到关键的调节基因，以确定培养和压力 VICRKX系统可能会感知的条件。遗传筛选和选择将用于 确定VICRKX系统感知的可能的信号和这些毒力的其他调节方式 因子基因。目标IV。我们将使用生化和遗传方法来确定VICX的作用 VICRKX信号转导中的第三个成分和假定的VICK磷酸酶活性。我们将确定 （VICX中的3-内酰胺倍折中的（3-内酰胺倍）是否在传感其他信号中起作用。AIM V.我们将使用 生化和微阵列方法，以确定遗漏的VICRKX法规的新成员 先前的研究。该赠款将提供有关重要细胞调节的基本新知识 严重的人类病原体中的壁生物合成和毒力因子基因。它将提供有关的见解 VICRKX系统在肺炎球菌和其他可能的其他可能的信号转导机制 链球菌的物种在细胞质和细胞表面之间进行通信。了解 VICRKX信号转导的异常特征将扩展TCS调节的范式。最后，基因 VICRKX调节中的产品是未来抗生素和疫苗开发的有前途的表面靶标。