Functions of the Essential Pneumococcal VicRKX Regulon

肺炎球菌必需 VicRKX 调节子的功能

• 批准号: 7046652
• 负责人: MALCOLM E. WINKLER
• 金额: $ 36.38万
• 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/7046652
• 关键词: DNA footprinting; Streptococcus pneumoniae; bacterial genetics; bacterial proteins; biological signal transduction; cell wall; chromatin immunoprecipitation; genetic regulation; genetic regulatory element; genetic screening; laboratory mouse; microarray technology; microorganism culture; microorganism metabolism; mutant; phosphorylation; protein binding; protein structure function; transcription factor; virulence

DESCRIPTION (provided by applicant): 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 beta-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.

描述（由申请方提供）：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。肺炎球菌的毒力需要基本的VicRK双组分系统（TCS）及其相关的第三组分VicX。该提案的长期目标是确定VicRKX系统用于调节编码必需胞壁蛋白生物合成酶的基因和肺炎球菌细胞表面上已建立的毒力因子的信号转导途径。我们的新结果表明，这种调节是直接的，并通过VicR反应调节因子（RR）的磷酸化介导。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信号的VicRKX系统，可能是由VicK组氨酸激酶（HK），它缺乏一个细胞外传感域，但含有PAS结构域，或其他磷酰基基团的捐助者。五个具体的目标将在这个五年的资助实现：目标一，我们将表征磷酸化VicR的结合，并使用体外生物化学方法在关键调节子基因成员的启动子区域产生的转录激活。Aim II.我们将确定VicRKX蛋白的表达水平和vicRKX突变体的毒力特性，以了解为什么VicK HK在S. pneumoniae在培养物中生长，但对毒力是必需的。我们将使用遗传方法来确定在VicK HK不存在的情况下，哪些其他供体磷酸化VicR RR。Aim III.我们将使用生物化学方法来确定VicK HK所感知的信号以及VicK HK是否具有VicR-P磷酸酶活性。我们将构建lacZ报告基因融合的关键调节子基因，以确定文化和应力条件下，可能会被VicRKX系统。遗传筛选和选择将用于识别VicRKX系统和这些毒力因子基因的其他调节模式所感知的可能信号。目标四。我们将使用生物化学和遗传学的方法来确定VicX的第三组分和推定的VicK磷酸酶活性在VicRKX信号转导中的作用。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。我们将使用生物化学和微阵列方法来确定VicRKX调节子的新成员，这些成员在以前的研究中被遗漏。该基金将提供有关严重人类病原体中重要细胞壁生物合成和毒力因子基因调控的基本新知识。它将提供对VicRKX系统在肺炎球菌和可能的其他链球菌物种中用于细胞质和细胞表面之间通信的多种信号转导机制的见解。理解VicRKX信号转导的不寻常特征将扩展TCS调节的范例。最后，VicRKX调节子中的基因产物是未来抗生素和疫苗开发的有希望的表面靶标。