Role of eukaryotic-type STK and PPPL in GAS pathogenesis

真核型 STK 和 PPPL 在 GAS 发病机制中的作用

• 批准号: 6905093
• 负责人: VIJAY PANCHOLI
• 金额: $ 16.14万
• 依托单位: PUBLIC HEALTH RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-03-15 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6905093
• 关键词: Streptococcus infection; Streptococcus pyogenes; bacterial genetics; bacterial proteins; bactericidal immunity; clinical research; gastrointestinal infection; genetic strain; human subject; laboratory mouse; microorganism growth; mutant; neutrophil; opsonin; phagocytosis; phosphoprotein phosphatase; protein structure function; respiratory infections; scanning electron microscopy; serine threonine protein kinase; transmission electron microscopy; virulence

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.

描述（由申请人提供）：A组链球菌（GAS，S.化脓性链球菌）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。 我们之前已经报道过GAS与宿主细胞相互作用后会引起细胞内信号传导并调节咽细胞中蛋白质的磷酸化。 原核生物中存在真核型丝氨酸/苏氨酸激酶（ESTK）和蛋白磷酸酶（ESTP），它们在原核生物代谢过程和信号转导途径中的调节作用已被认识。然而，它们的确切功能尚不清楚，因为在大多数生物中，几个基因编码结构相似的蛋白质。GAS基因组序列分析还揭示了存在各自编码ESTK和ESTP的单个基因，其分别被注释为STK和pppL。关于ESTK和/或ESTP在革兰氏阳性菌致病机制中的作用的信息非常有限。特别是，STK和pppL在GAS发病机制中的作用尚不清楚。在这个建议中，我们已经确定，其特征在于和克隆的STK和pppL特异性基因，产生相应的重组蛋白，并产生STK和pppL特异性的多克隆抗体。突变株，缺乏STK或其C-末端的外部结构域，表现出显着的变化，表面蛋白的表达和细胞分裂的轮廓，所揭示的扫描和透射电子显微镜。根据这些结果，我们推测STK和pppL在GAS发病机制中起重要作用。我们提出了几种策略来创建和表征突变株缺乏STK，pppL的激酶结构域的表达，以及STK和pppL的遗传，生化和免疫学分析。 在后续的特定目的中，我们将使用体外试验检测所有突变菌株的毒力，以检测GAS粘附和侵入咽细胞以及人中性粒细胞对其的吞噬作用，并通过特异性抗体介导的粘附抑制和调理吞噬试验确认其毒力潜力。这些体外研究结果将通过体内小鼠腹腔内和鼻咽感染模型进行验证。从这项探索性资助中获得的结果将有助于了解这些新型信号分子在细菌发病机制中的作用，特别是GAS发病机制。