Metal-dependent phosphohydrolase activity of CvfA from Streptococcus pyogenes

描述(申请人提供)：保守的毒力因子A(CvfA)含有金属依赖的磷酸水解酶结构域，调节化脓性链球菌在低营养条件下糖酵解酶和毒力因子的表达。A组链球菌(GAS)与多种人类疾病有关，包括猩红热、脓疱疮、咽炎、坏死性筋膜炎、链球菌中毒性休克综合征以及风湿热的链球菌后遗症。依赖于生长阶段的蛋白表达调控允许适应不同的宿主组织，并代表着毒力因子表达变化所反映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特异性和营养依赖的信号反应来了解基因调控的机制。我们以前的研究表明，CvfA与糖酵解酶烯醇化酶有关，这可能是一种兼职的CvfA活性的营养敏感调节因子。我们还发现，在低碳水化合物的介质中，CvfA下调毒力因子M蛋白、链激酶和cAMP因子的表达，而上调分泌的蛋白酶SpeB的表达。初步研究表明，纯化的重组CvfA蛋白具有特异性切割M蛋白的mRNA转录物的内切酶活性，而对RecA的mRNA转录物没有影响。我们的工作假设是，CvfA是一种序列特异性内切酶，它利用RNA结合的KH结构域来靶向mRNA转录，使其被含有组氨酸-天冬氨酸(HD)的磷酸水解酶结构域切割，从而通过RNA降解来调节基因的表达。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，3)确定HD结构域的金属依赖的催化活性和结构。我们的方法结合了结构研究和功能分析，包括核磁共振波谱研究KH和HD结构域结构，催化分析确定磷酸水解酶活性的机制，mRNA衰变分析解释营养信号反应，mRNA切割分析和体外筛选确定CvfA的序列特异性。我们的结果的影响将导致新的治疗策略，遵循三种潜在的方法：1)干扰KH结构域的RNA识别，2)下调营养信号通路，以及3)基于催化分析确定的先导化合物开发磷酸水解酶活性的抑制剂。 公共卫生相关性：化脓性链球菌是一种重要的人类病原体，由于尚不存在安全的疫苗，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，以响应养分的可获得性。我们将研究ORDR中CvfA的结构和细胞功能，以了解毒力控制的机制。这将使我们能够根据我们对CvfA的了解开始开发针对链球菌疾病的治疗措施。

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.