Functions of the Essential Pneumococcal VicRKX Regulon

描述(申请人提供)：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，正在对抗生素产生耐药性。肺炎球菌毒力所必需的VicRK双组分系统(TCS)及其相关的第三组分VicX是必需的。这项建议的长期目标是确定VicRKX系统用来调节编码一种重要的粘菌素生物合成酶的基因的信号转导途径，并确定肺炎球菌细胞表面的毒力因子。我们的新结果表明，这种调节是直接的，并通过VICR反应调节因子(RR)的磷酸化来介导。其他新的结果表明，细胞壁生物合成缺陷可能会产生由VicRKX系统感应的代谢信号，可能是通过缺乏细胞外传感结构域但含有PAS结构域的维克组氨酸激酶(HK)或其他磷酸基供体。这项为期五年的拨款将实现五个具体目标：目的I，我们将利用体外生化方法表征磷酸化的VICR结合以及由此产生的关键调节子基因启动子区域的转录激活。目的II.我们将测定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不是肺炎链球菌在培养中所必需的，而是毒力所必需的。我们将使用遗传方法来确定在VICK HK不存在的情况下，哪些其他捐赠者将VICR RR磷酸化。目的III.我们将使用生化方法来确定VICK HK感受到的信号(S)以及VICK HK是否具有VICR-P磷酸酶活性。我们将构建LacZ报告基因与关键调节子基因的融合，以确定VicRKX系统可能感知的培养和胁迫条件。基因筛选和选择将被用来识别VicRKX系统和这些毒力因子基因的其他调节模式可能感受到的信号。目的IV.我们将使用生化和遗传学方法来确定VicX第三组分和可能的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方面发挥作用。目的V.我们将使用生化和微阵列方法来确定先前研究中遗漏的VicRKX调节子的新成员。这项拨款将为人类严重病原体的重要细胞壁生物合成和毒力因子基因的调控提供基本的新知识。它将深入了解VicRKX系统在肺炎球菌和可能其他种类的链球菌中用来在细胞质和细胞表面之间通信的多种信号转导机制。了解VicRKX信号转导的异常特征将扩展TCS调控的范式。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点。

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.