Role of a novel signal transduction pathway in GBS

描述(申请人提供)：革兰氏阳性病原体无乳链球菌(B组链球菌，GBS)是人类新生儿肺炎、败血症和脑膜炎的主要原因。GBS也是免疫低下成人的一种新的病原体。我们最近在GBS中发现并鉴定了一个新的真核型丝氨酸/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的分离具有多效性，表明该信号转导途径在调节多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蛋白的可逆磷酸化是必不可少的。通过质谱分析，我们确定其中一个靶标是依赖于锰的无机焦磷酸酶(PPAC)。焦磷酸酶对调节细胞内的生物合成反应至关重要。根据我们的结果，我们假设这一信号转导途径及其靶点的翻译后修饰对于GBS的正常细胞功能至关重要。我们将结合分子、生化和蛋白质组学的方法来阐明这一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，我们将确定和表征这一途径的上游和下游靶点。我们将利用现代蛋白质组学技术，如液相色谱和质谱学，来确定这一信号转导途径的其他关键靶点。我们将进行缺失分析和蛋白质交联研究，以确定与Stk1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的已鉴定生理底物PPAC的功能表征。在目标3中，我们将构建PPAC和目标1中确定的该途径的其他几个调控靶点的突变，以评估它们在GBS生长和生存中的作用。我们预计，包括PPAC在内的一些基因将对GBS的生长至关重要。由于突变的非必需基因不能存活，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。总之，这些研究将确定这一信号转导途径的生物学意义，并导致识别GBS的新靶点，这可能为了解其作为抗菌靶点的潜力提供见解。

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.