Role of eukaryotic-type STK and PPPL in GAS pathogenesis

描述(申请人提供)：A组链球菌(GAS，化脓性链球菌)可引起多种人类疾病，从轻度咽炎和皮肤病到严重坏死性筋膜炎、中毒性休克综合征和自身免疫性疾病。我们早些时候已经报道，GAS在与宿主细胞相互作用时，会激活细胞内信号事件，并调节咽部细胞中的蛋白质磷酸化。真核型丝氨酸/苏氨酸激酶(ESTK)和蛋白磷酸酶(ESTP)在原核生物中的存在，以及它们在原核生物代谢过程和信号转导途径中的调节作用，近年来已被认识到。然而，它们的确切功能尚不清楚，因为在大多数生物体中，有几个基因编码结构相似的蛋白质。GAS基因组序列分析还显示，ESTK和ESTP各有一个编码基因，分别被注释为STK和PPPL。关于ESTK和/或ESTP在革兰氏阳性细菌发病机制中的作用的信息极其有限。特别是，STK和PPPL在GAS发病机制中的作用尚不清楚。在这个方案中，我们鉴定、鉴定和克隆了STK和PPPL特异性基因，产生了相应的重组蛋白，并制备了STK和PPPL特异性多克隆抗体。扫描和透射电子显微镜显示，缺乏STK或其C末端外区的突变菌株，其表面蛋白的表达和细胞分裂谱发生了显著变化。根据这些结果，我们推测STK和PPPL在GAS的发病机制中起重要作用。我们提出了几种策略，通过遗传、生化和免疫学分析来创造和鉴定缺乏STK、PPPL以及STK和PPPL激活域表达的突变株。在随后的特定目标中，我们将使用体外气体黏附和侵袭咽部细胞以及人中性粒细胞吞噬能力的实验来测试所有突变菌株的毒力，并通过特异性抗体介导的黏附抑制和吞噬细胞实验来确认它们的毒力潜力。这些体外发现将通过体内小鼠腹膜和鼻咽部感染模型进行验证。这项探索性拨款的结果将有助于理解这些新的信号分子在细菌发病机制中的作用，特别是在GAS发病机制中的作用。

DESCRIPTION (provided by applicant): Group A Streptococcus (GAS, S. pyogenes) causes a variety of human diseases ranging from mild pharyngitis and skin diseases to severe necrotizing fasciits, toxic shock syndrome, and autoimmune diseases. We have reported earlier that GAS upon interaction with host cells invokes intracellular signaling events and regulates protein phosphoryaltion in pharyngeal cells. The presence of the eukaryotic-type Ser/Thr kinase (ESTK) and protein phosphatase (ESTP) in prokaryotes and their functional importance in terms of regulatory roles in prokaryotic metabolic processes and signal transduction pathways have been recently recognized. However, their precise function is not known, since in most organisms, several genes encode for structurally similar proteins. GAS genome sequence analyses have also revealed the presence of a single gene each encoding for ESTK and ESTP, which are annotated as STK and pppL respectively. The information on the role of ESTK and/or ESTP in gram-positive bacterial pathogenesis is extremely limited. In particular, the role of STK and pppL in GAS pathogenesis is not known. In this proposal, we have identified, characterized and cloned STK and pppL specific genes, produced corresponding recombinant proteins and generated STK and pppL-specific polyclonal antibodies. Mutants strains, lacking STK or its C-terminal outer domains, exhibited remarkable changes in the expression of surface proteins and cell division profiles as revealed by scanning and transmission electron microscopy. With these results, we hypothesize that STK and pppL play a significant role in GAS pathogenesis. We propose several strategies to create and characterize mutant strains lacking the expression of kinase domain of STK, pppL, and both STK and pppL by genetic, biochemical and immunological analyses. In subsequent Specific Aims, we will test virulence of all the mutant strains using in vitro assays for GAS adherence to and invasion of pharyngeal cells and its phagocytosis by human neutrophils and confirm their virulence potential by specific antibody-mediated adherence inhibition and opsonophagocytosis assays. These in vitro findings will be validated with in vivo mouse intraperitoneal and nasopharyngeal infection models. The results obtained from this exploratory grant will allow to understand the roles of these novel signaling molecules in bacterial pathogenesis in general and GAS pathogenesis in particular.