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Metal-dependent phosphohydrolase activity of CvfA from Streptococcus pyogenes

化脓链球菌 CvfA 的金属依赖性磷酸水解酶活性

基本信息

批准号：
8957072
负责人：
Kyu Hong Cho
金额：
$ 30.66万
依托单位：
IOWA STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-08-01 至 2017-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8957072
关键词：
Metal dependent phosphohydrolase activity CvfA

项目摘要

DESCRIPTION (provided by applicant): Conserved virulence factor A (CvfA) contains a metal-dependent phosphohydrolase domain and regulates the expression of glycolytic enzymes and virulence factors of Streptococcus pyogenes in response to low nutrient conditions. Group A Streptococcus (GAS) is associated with a broad range of human diseases including scarlet fever, impetigo, pharyngitis, necrotizing fasciitis, streptococcal toxic shock syndrome and the post- streptococcal sequelae of rheumatic fever. The growth phase dependent regulation of protein expression allows for adaptation to various host tissues and represents a potential switch in the mode of infection reflected in the change in virulence factor expression. Our overall goal is to understand the mechanism of gene regulation by defining the catalytic activity, the RNA specificity and the nutrient-dependent signaling response of CvfA. Our previous studies indicated that CvfA is associated with the glycolytic enzyme, enolase, which may be moonlighting as a nutrient-sensing regulator of CvfA activity. We also showed that in low-carbohydrate media, CvfA down-regulates the expression of the virulence factors M protein, streptokinase and CAMP factor, while up-regulating expression of the secreted protease, SpeB. Preliminary studies show that purified recombinant CvfA protein has endonuclease activity that specifically cleaves the mRNA transcript of M protein, while showing no effects on the mRNA transcript of RecA. Our working hypothesis is that CvfA is a sequence- specific endonuclease that utilizes an RNA-binding KH domain to target mRNA transcripts for cleavage by the histidine-aspartate containing (HD) phosphohydrolase domain resulting in regulation of gene expression through RNA degradation. Our specific aims are to: 1) determine the structure and RNA binding specificity of the KH domain, 2) identify the mRNA substrates of CvfA and define the nutrient dependent regulatory role of CvfA, and 3) determine the metal-dependent catalytic activity and structure of the HD domain. Our approach combines structural studies and functional assays including NMR spectroscopy to study the KH and HD domain structures, catalytic assays to define the mechanism of phosphohydrolase activity, mRNA decay assays to elucidate the nutrient signaling response, mRNA cleavage assays and in vitro selection to define the sequence specificity of CvfA. The impact of our results will lead to new therapeutic strategies against streptococcal infections following three potential approaches: 1) interference with RNA recognition by the KH domain, 2) down regulation of nutrient signaling pathways, and 3) developing inhibitors of phosphohydrolase activity based on lead compounds identified through catalytic assays. PUBLIC HEALTH RELEVANCE: Streptococcus pyogenes is an important human pathogen against which new therapeutic methods must be developed urgently since safe vaccines do not exist. CvfA influences the expression of genes controlling virulence and nutrient utilization in response to nutrient availability. We will study the structure and cellular function of CvfA in ordr to understand the mechanism of the virulence control. This will allow us to begin developing therapeutic measures against streptococcal diseases based on our knowledge of CvfA.

描述（由申请方提供）：保守毒力因子A（CvfA）含有金属依赖性磷酸水解酶结构域，并调节酿脓链球菌糖酵解酶和毒力因子在低营养条件下的表达。A组链球菌（GAS）与广泛的人类疾病相关，包括猩红热、脓疱病、咽炎、坏死性筋膜炎、链球菌中毒性休克综合征和风湿热的链球菌感染后后遗症。蛋白表达的生长期依赖性调节允许适应各种宿主组织，并代表了反映在毒力因子表达变化中的感染模式的潜在转换。我们的整体目的是通过定义CvfA的催化活性、RNA特异性和营养依赖性信号应答来理解基因调控机制。我们以前的研究表明，CvfA与糖酵解酶，烯醇化酶，这可能是兼职作为CvfA活性的营养传感调节器。我们还发现，在低碳水化合物培养基中，CvfA下调毒力因子M蛋白、链激酶和CAMP因子的表达，同时上调分泌蛋白酶SpeB的表达。初步研究表明，纯化的重组CvfA蛋白具有内切酶活性，特异性切割M蛋白的mRNA转录本，而对RecA的mRNA转录本没有影响。我们的工作假设是CvfA是一种序列特异性内切核酸酶，其利用RNA结合KH结构域靶向mRNA转录物，用于被含组氨酸-天冬氨酸（HD）的磷酸水解酶结构域切割，导致通过RNA降解调节基因表达。我们的具体目标是：1）确定KH结构域的结构和RNA结合特异性，2）鉴定CvfA的mRNA底物并确定CvfA的营养依赖性调节作用，以及3）确定HD结构域的金属依赖性催化活性和结构。我们的方法结合了结构研究和功能测定，包括NMR光谱研究KH和HD结构域的结构，催化测定，以确定磷酸水解酶活性的机制，mRNA衰变测定，以阐明营养信号传导反应，mRNA裂解测定和体外选择，以确定CvfA的序列特异性。我们的研究结果的影响将导致新的治疗策略对链球菌感染以下三个潜在的方法：1）干扰RNA识别的KH结构域，2）下调营养信号传导途径，和3）开发磷酸水解酶活性的抑制剂的基础上通过催化试验确定的先导化合物。公共卫生关系：化脓性链球菌是一种重要的人类病原体，由于没有安全的疫苗，因此迫切需要开发新的治疗方法。CvfA影响控制毒力和养分利用的基因的表达，以响应养分的可用性。我们将研究CvfA的结构和细胞功能，以了解其毒力控制机制。这将使我们能够开始根据我们对CvfA的了解开发针对链球菌疾病的治疗措施。