Functions of the Essential Pneumococcal VicRKX Regulon

描述（由申请人提供）：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，正在产生抗生素耐药性。必不可少的VicRK双组分系统（TCS）及其相关的第三组分VicX是肺炎球菌毒力所必需的。该建议的长期目标是确定VicRKX系统用于调节编码必需小鼠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，并且是由VicR反应调节因子（RR）的磷酸化介导的。其他新的研究结果表明，细胞壁生物合成缺陷可能产生由VicRKX系统感知的代谢信号，可能是由缺乏细胞外感知结构域但含有PAS结构域的vik组氨酸激酶（HK）或其他磷酸化基供体感知的代谢信号。在这个为期五年的资助中，我们将实现五个特定目标：目标一，我们将使用体外生化方法表征磷酸化的VicR的结合以及关键调控基因成员启动子区域的转录激活。目的二世。我们将确定VicRKX蛋白的表达水平和VicRKX突变体的毒力特性，以了解为什么维克HK在培养的肺炎链球菌生长中不是必需的，但在毒力中是必需的。我们将使用遗传方法来确定哪些其他供体在没有维克HK的情况下磷酸化了维克RR。第三目标。我们将使用生化方法来确定VicK HK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报告基因与关键调控基因的融合体，以确定可能被VicRKX系统感知的培养和胁迫条件。遗传筛选和选择将用于识别由VicRKX系统感知的可能信号以及这些毒力因子基因的其他调节模式。目的四：我们将使用生化和遗传方法来确定VicX第三组分和假定的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。目的五：我们将使用生化和微阵列方法来确定在以前的研究中遗漏的VicRKX调控的新成员。这项资助将提供有关重要细胞壁生物合成和严重人类病原体毒力因子基因调控的基本新知识。它将为肺炎球菌和可能的其他种类链球菌中VicRKX系统用于细胞质和细胞表面之间通信的信号转导的多种机制提供见解。了解VicRKX信号转导的不寻常特征将扩展TCS调控的范式。最后，VicRKX调控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.