Role of a novel signal transduction pathway in GBS

一种新的信号转导途径在 GBS 中的作用

• 批准号: 7008171
• 负责人: CRAIG E. RUBENS
• 金额: $ 30.27万
• 依托单位: SEATTLE CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-30 至 2008-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7008171
• 关键词: Streptococcus agalactiae; binding sites; biological signal transduction; clinical research; crosslink; enzyme mechanism; gene induction /repression; gene mutation; genetic regulation; host organism interaction; human tissue; laboratory rat; liquid chromatography; manganese; mass spectrometry; microorganism growth; phosphoprotein phosphatase; posttranslational modifications; protein structure function; serine threonine protein kinase; site directed mutagenesis

DESCRIPTION (provided by applicant): The gram-positive pathogen Streptococcus agalactiae (group B streptococci, GBS) is the principal cause of human neonatal pneumonia, sepsis and meningitis. GBS is also an emerging pathogen of immunocompromised adults. We recently identified and characterized a novel eukaryotic-type serine/threonine protein kinase (Stk1) and its cognate phosphatase (Stp1) in GBS. Mutants of this signal transduction pathway exhibited pleiotropic effects on cell growth, virulence and segregation of GBS, indicating the importance of this pathway in the regulation of various cellular processes. In vitro phosphorylation studies revealed that these enzymes are essential for reversible phosphorylation of many GBS proteins. Using mass spectrometric analysis, we identified one of these targets as a anganese-dependent inorganic pyrophosphatase (PpaC). Pyrophophatases are critical for regulation of biosynthetic reactions in the cell. Based on our results, we hypothesize that this signal transduction pathway and post-translational modification of its targets are crucial for normal cellular functions in GBS. A combination of molecular, biochemical and proteomic approaches will be used to elucidate the role of this signal transduction pathway and its physiological substrates in growth and virulence of GBS. In aim 1, we will identify and characterize the upstream and downstream targets of this pathway. We will utilize modem proteomic techniques such as liquid chromatography and mass spectrometry to identify other key targets of this signal transduction pathway. We will perform deletion analysis and protein cross-linking studies, to identify proteins that bind to and activate Stk1. In Aim 2, we will complete functional characterization of the identified physiological substrate of this signal transduction pathway, PpaC. In Aim 3, we will construct mutations in ppaC and a few other regulated targets of this pathway, identified in aim 1, to assess their role in growth and survival of GBS. We anticipate that some of these genes including ppaC will be essential for GBS growth. As mutants inessential genes are not viable, we will use the modem RNA interference technology to evaluate their role in GBS growth and survival. Collectively, these studies will determine the biological significance of this signal transduction pathway and lead to the identification of novel targets of GBS, which may provide insights into their potential as antimicrobial targets.

描述（由申请人提供）：革兰氏阳性病原体无乳链球菌（B 族链球菌，GBS）是人类新生儿肺炎、败血症和脑膜炎的主要原因。 GBS 也是免疫功能低下成人的一种新病原体。我们最近在 GBS 中鉴定并表征了一种新型真核型丝氨酸/苏氨酸蛋白激酶 (Stk1) 及其同源磷酸酶 (Stp1)。该信号转导途径的突变体对细胞生长、毒力和 GBS 分离表现出多效性，表明该途径在调节各种细胞过程中的重要性。体外磷酸化研究表明，这些酶对于许多 GBS 蛋白的可逆磷酸化至关重要。通过质谱分析，我们确定了其中一个靶标为锰依赖性无机焦磷酸酶 (PpaC)。焦磷酸酶对于细胞生物合成反应的调节至关重要。根据我们的结果，我们假设这种信号转导途径及其靶标的翻译后修饰对于 GBS 的正常细胞功能至关重要。将结合分子、生物化学和蛋白质组学方法来阐明该信号转导途径及其生理底物在 GBS 生长和毒力中的作用。在目标 1 中，我们将确定并描述该途径的上游和下游目标。我们将利用现代蛋白质组学技术（例如液相色谱法和质谱法）来识别该信号转导途径的其他关键靶标。我们将进行缺失分析和蛋白质交联研究，以鉴定结合并激活 Stk1 的蛋白质。在目标 2 中，我们将完成该信号转导途径已识别的生理底物 PpaC 的功能表征。在目标 3 中，我们将在目标 1 中确定的 ppaC 和该通路的其他一些调控靶标中构建突变，以评估它们在 GBS 生长和存活中的作用。我们预计其中一些基因（包括 ppaC）对于 GBS 生长至关重要。由于突变体非必需基因无法存活，我们将使用现代RNA干扰技术来评估它们在GBS生长和存活中的作用。总的来说，这些研究将确定该信号转导途径的生物学意义，并确定 GBS 的新靶点，这可能有助于深入了解它们作为抗菌靶点的潜力。