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

• 批准号: 8289909
• 负责人: Kyu Hong Cho
• 金额: $ 0.49万
• 依托单位: SOUTHERN ILLINOIS UNIVERSITY CARBONDALE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-08-01 至 2014-05-20
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8289909
• 关键词: Acute Glomerulonephritis; Aspartate; Bacillus subtilis; Binding; Biological Assay; Boxing; Carbohydrates; Cell physiology; Chemicals; Cleaved cell; Dependence; Disease; Down-Regulation; Elements; Enzymes; Gene Expression; Gene Expression Regulation; Genes; Goals; Growth; Guanosine Tetraphosphate; Histidine; Human; Impetigo; In Vitro; Infection; Ions; KH Domain; Knowledge; Lead; Maps; Measures; Mediating; Messenger RNA; Metals; Methods; Molecular; N-terminal; NMR Spectroscopy; Necrotizing fasciitis; Nutrient; Nutritional; Orthologous Gene; Pathogenesis; Peptide Hydrolases; Pharyngitis; Phase; Phosphoric Monoester Hydrolases; Proteins; RNA; RNA Binding; RNA Degradation; RNA Sequences; Recombinants; Regulation; Rheumatic Fever; Ribonucleoproteins; Role; Scarlet Fever; Signal Pathway; Signal Transduction; Specificity; Streptococcal Infections; Streptococcus pyogenes; Streptokinase; Stress; Structure; Structure-Activity Relationship; Testing; Therapeutic; Tissues; Toxic Shock Syndrome; Transcript; Translational Regulation; Transmembrane Domain; Vaccines; Virulence; Virulence Factors; Work; base; cis acting element; design; detection of nutrient; endonuclease; enolase; factor A; gene repression; human disease; in vitro Assay; in vivo; inhibitor/antagonist; mRNA Decay; multiple myeloma M Protein; novel therapeutics; pathogen; phosphoric diester hydrolase; protein expression; response

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（由申请人提供）：保守毒力因子 A (CvfA) 含有金属依赖性磷酸水解酶结构域，并调节化脓性链球菌糖酵解酶和毒力因子的表达，以响应低营养条件。 A 族链球菌 (GAS) 与多种人类疾病有关，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链球菌感染后后遗症。蛋白质表达的生长阶段依赖性调节允许适应各种宿主组织，并代表毒力因子表达变化所反映的感染模式的潜在转变。我们的整体 目标是通过定义 CvfA 的催化活性、RNA 特异性和营养依赖性信号反应来了解基因调控机制。我们之前的研究表明，CvfA 与糖酵解酶、烯醇化酶有关，后者可能兼职作为 CvfA 活性的营养感应调节剂。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蛋白具有核酸内切酶活性，可以特异性切割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 是一种序列特异性核酸内切酶，它利用 RNA 结合 KH 结构域来靶向 mRNA 转录本，并通过含有组氨酸天冬氨酸的 (HD) 磷酸水解酶结构域进行切割，从而通过 RNA 降解来调节基因表达。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) 识别 CvfA 的 mRNA 底物并定义 CvfA 的营养依赖性调节作用，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，包括用于研究 KH 和 HD 结构域结构的 NMR 光谱、用于定义磷酸水解酶活性机制的催化测定、用于阐明营养信号反应的 mRNA 衰减测定、用于定义 CvfA 序列特异性的 mRNA 裂解测定和体外选择。我们的结果的影响将导致针对链球菌感染的新治疗策略，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）基于通过催化测定鉴定的先导化合物开发磷酸水解酶活性抑制剂。 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，因此必须紧急开发新的治疗方法。 CvfA 影响控制毒力和营养利用以响应营养可用性的基因的表达。我们将研究CvfA的结构和细胞功能，以便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。