Signal Transduction by Essential VicRKX in Pneumococcus

肺炎球菌中必需 VicRKX 的信号转导

• 批准号: 7335582
• 负责人: MALCOLM E. WINKLER
• 金额: $ 35.75万
• 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 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7335582
• 关键词: Affect; Anabolism; Animal Model; Animals; Antibiotic Resistance; Antibiotics; Bacteria; Binding; Biochemical; Biochemical Genetics; Biochemical Reaction; Cell Wall; Cell surface; Cells; Condition; 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; Rate; 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， 正在产生抗生素耐药性基本的VicRK双组分系统（TCS）及其相关 第三组分VicX是肺炎球菌毒力所必需的。该提案的长期目标是 确定VicRKX系统用于调节基因编码的信号转导途径， 必需的胞壁素生物合成酶和肺炎球菌细胞表面上已建立的毒力因子。 我们的新结果表明，这种调节是直接的，并通过VicR反应的磷酸化介导。 调节器（RR）。其他新的结果表明，有缺陷的细胞壁生物合成可能会产生代谢产物， 由VicRKX系统感知的信号，可能是由VicK组氨酸激酶（HK）感知的，其缺乏一个 细胞外传感结构域，但含有PAS结构域，或通过其他磷酰基供体。五个具体 本研究计划在五年内完成以下目标：目标一，我们将研究磷酸化VicR的结合特性 以及在关键调节子基因成员的启动子区域处产生的转录激活， 生物化学方法Aim II.我们将确定VicRKX蛋白的表达水平和毒力 vicRKX突变体的性质，以理解为什么VicK HK在S.肺炎菌生长在 文化，但需要毒力。我们将用遗传学方法来确定其他捐赠者 在不存在VicK HK的情况下使VicR RR磷酸化。Aim III.我们将使用生物化学方法， 确定由VicK HK感测的信号以及VicK HK是否具有VicR-P磷酸酶 活动我们将构建lacZ报告融合关键调节子基因，以确定文化和压力 VicRKX系统可能感测到的条件。遗传筛选和选择将用于 确定VicRKX系统和这些毒力的其他调节模式所感知的可能信号 因子基因。目标四。我们将使用生物化学和遗传学方法来确定VicX的作用 第三组分和VicRKX信号转导中的推定VicK磷酸酶活性。我们将确定 VicX中的β-内酰胺酶折叠是否在感知额外信号中起作用。目标五：我们将使用 生物化学和微阵列方法，以确定新成员的VicRKX调节子，错过了在 以前的研究。这项资助将提供有关重要细胞调节的基本新知识， 壁生物合成和毒力因子基因在一个严重的人类病原体。它将提供深入了解 VicRKX系统在肺炎球菌和可能的其他细菌中使用的多种信号转导机制 在细胞质和细胞表面之间进行通讯的链霉菌。了解 VicRKX信号转导的不寻常特征将扩展TCS调节的范例。最后，吉恩 VicRKX调节子中的产物是未来抗生素和疫苗开发的有希望的表面靶标。