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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：
7335582
负责人：
MALCOLM E. WINKLER
金额：
$ 35.75万
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 2010-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7335582
关键词：
Affect Anabolism Animal Model Animals Antibiotic Resistance Antibiotics Bacteria Binding Biochemical Biochemical Genetics Biochemical Reaction Cell Wall Cell surface Cells Condition 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 Rate 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