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。肺炎球菌毒力需要必需的 VicRK 二组分系统 (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的胞壁质生物合成酶的基因以及在肺炎球菌细胞表面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，并由 VicR 反应调节器 (RR) 的磷酸化介导。其他新结果表明，有缺陷的细胞壁生物合成可能会产生由 VicRKX 系统感知的代谢信号，可能是由 VicK 组氨酸激酶 (HK)（缺乏细胞外感知结构域但包含 PAS 结构域）或其他磷酰基供体感知的。这项为期五年的资助将实现五个具体目标： 目标 I，我们将使用体外生化方法表征磷酸化 VicR 的结合以及在关键调节子基因成员的启动子区域产生的转录激活。目标二。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 在培养的肺炎链球菌中不是必需的，但却是毒力所必需的。我们将使用遗传方法来确定在没有 VicK HK 的情况下哪些其他供体使 VicR RR 磷酸化。目标三。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。我们将构建与关键调节子基因的 lacZ 报告基因融合体，以确定 VicRKX 系统可能感知的培养和应激条件。遗传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因子基因的其他调节模式。目标四。我们将使用生化和遗传学方法来确定 VicX 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附加信号中发挥作用。目标 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。这笔赠款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节的基础新知识。它将深入了解 VicRKX 系统在肺炎球菌和可能的其他种类链球菌中使用的多种信号转导机制，以在细胞质和细胞表面之间进行通讯。了解 VicRKX 信号转导的不寻常特征将扩展 TCS 调节的范式。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。