Signal Transduction by Essential VicRKX in Pneumococcus

肺炎球菌中必需 VicRKX 的信号转导

基本信息

批准号：
7748997
负责人：
MALCOLM E. WINKLER
金额：
$ 35.36万
依托单位：
TRUSTEES OF INDIANA UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2006
资助国家：
美国
起止时间：
2006-01-01 至 2011-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7748997
关键词：
Affect Anabolism Animal Model Animals Antibiotic Resistance Antibiotics Bacteria Binding Biochemical Biochemical Genetics Biochemical Reaction Cell Wall Cell surface Cells Cytoplasm DNA Data Enzymes Flavin Mononucleotide Fluorescence Anisotropy Future Gene Expression Gene Targeting Genes Genetic Genetic Screening Genetic Transcription Goals Grant Growth Human In Vitro Infection Knowledge LacZ Genes Lactamase Measures Mediating Membrane Proteins Metabolic Metals Methods Nature Peptidoglycan Phosphoric Monoester Hydrolases Phosphorylation Physiological Physiology Play Promoter Regions Property Proteins Pyruvate Oxidase Reaction Regulation Regulon Reporter Research Personnel Resistance development Role Running Screening procedure Signal Transduction Signal Transduction Pathway Site Streptococcus Streptococcus pneumoniae Stress Surface System Testing Time Transcriptional Activation Transcriptional Regulation Transferase Virulence Virulence Factors acetyl phosphate base extracellular feeding genetic selection in vivo insight member mutant pathogen programs protein-histidine kinase research study respiratory response small molecule vaccine development yeast two hybrid system

项目摘要

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.

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，正在形成抗生素耐药性。基本的 VicRK 二元系统 (TCS) 及其相关第三种成分 VicX 是肺炎球菌毒力所必需的。该提案的长期目标是确定 VicRKX 系统用于调节编码基因的信号转导途径必需的胞壁质生物合成酶和肺炎球菌细胞表面已建立的毒力因子。我们的新结果表明，这种调节是直接的，并由 VicR 反应的磷酸化介导调节器（RR）。其他新结果表明，有缺陷的细胞壁生物合成可能会产生代谢 VicRKX 系统感知的信号，可能是由 VicK 组氨酸激酶 (HK) 感知的，该激酶缺乏胞外传感结构域但包含 PAS 结构域，或由其他磷酰基供体组成。五具体这项为期五年的资助将实现的目标：目标 I，我们将表征磷酸化 VicR 的结合以及在体外使用在关键调节子基因成员的启动子区域产生的转录激活生化方法。目标二。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 在肺炎链球菌生长中不是必需的文化，但需要毒力。我们将使用遗传方法来确定哪些其他捐赠者在没有 VicK HK 的情况下磷酸化 VicR RR。目标三。我们将使用生化方法确定 VicK HK 感应到的信号以及 VicK HK 是否具有 VicR-P 磷酸酶活动。我们将构建与关键调节子基因的 lacZ 报告基因融合体，以确定培养和应激 VicRKX 系统可能检测到的条件。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的其他调节模式因子基因。目标四。我们将使用生化和遗传学方法来确定 VicX 的作用 VicRKX 信号转导中的第三个成分和推定的 VicK 磷酸酶活性。我们将确定 VicX 中的 3-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 VicRKX 调节子的新成员，这些成员在以前的研究。这笔赠款将提供有关重要细胞调节的基础新知识严重人类病原体中的壁生物合成和毒力因子基因。它将提供有关 VicRKX 系统在肺炎球菌和其他可能的球菌中使用的多种信号转导机制链球菌种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，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目标。