Signal Transduction by Essential VicRKX in Pneumococcus

肺炎链球菌(简称肺炎球菌)是一种重要的革兰氏阳性人类呼吸道病原体。 正在产生抗生素抗药性。本质VicRK二组分系统(TCS)及其相关 第三组分VicX是肺炎球菌毒力所必需的。这项提议的长期目标是 确定VicRKX系统用于调节编码基因的信号转导途径 肺炎球菌细胞表面必需的粘菌素生物合成酶和已建立的毒力因子。 我们的新结果表明，这种调节是直接的，并且是通过vir反应的磷酸化来介导的。 调节器(RR)。其他新结果表明，细胞壁生物合成缺陷可能会产生新陈代谢 由VicRKX系统感应的信号，可能是通过维克组氨酸激酶(HK)，它缺乏一种 胞外感应域，但含有PAS结构域，或由其他磷酸基供体。五个具体 这项为期五年的拨款将实现以下目标：目标I，我们将表征磷酸化VICR的结合 以及由此产生的关键调节子基因成员启动子区域的转录激活 生化方法。目的II.我们将测定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不是肺炎链球菌生长所必需的 培养，但这是毒力所必需的。我们将使用基因方法来确定哪些其他捐赠者 在VICK HK不在的情况下将VICR RR磷酸化。目标三.我们将使用生化方法来 确定VICK HK感测到的信号(S)以及VICK HK是否具有VICR-P磷酸酶 活动。我们将构建LacZ报告基因与关键调控基因的融合，以确定文化和压力 VicRKX系统可能检测到的条件。基因筛选和选择将用于 识别VicRKX系统感知的可能信号以及这些毒力的其他调节模式 因子基因。目的IV.我们将使用生化和遗传学方法来确定VicX的作用 VicRKX信号转导中的第三组分和可能的VICK磷酸酶活性。我们将决定 VicX中的(3-内酰胺酶折叠)是否在感知额外信号方面发挥作用。Aim V.我们将使用 用生化和微阵列方法确定在 以前的研究。这项资助将提供关于重要细胞调控的基本新知识 一种严重的人类病原体的壁生物合成和毒力因子基因。它将提供对 VicRKX系统在肺炎球菌和其他类似疾病中使用的多种信号转导机制 链球菌的一种，在细胞质和细胞表面之间通信。了解 VicRKX信号转导的不寻常特征将扩展TCS调控的范式。最后，基因 VicRKX调节子中的产品是未来抗生素和疫苗开发的有希望的表面靶点。

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.