Epac1 Plays a Critical Role in Bacterial Adhesion during Rickettsioses

 描述（由申请人提供）：立克次体病是毁灭性的人类感染。流行性斑疹伤寒和落基山斑疹热（RMSF）是人类已知的两种最致命的感染。虽然立克次体感染可以通过适当的广谱抗生素治疗控制，如果早期诊断，高达20%的误诊或未经治疗和5%的治疗RMSF病例可能是致命的。此外，立克次体的高传染性和吸入后的严重病情使其成为潜在的生物恐怖主义威胁。然而，疫苗不是 可用于致死性立克次体病，并且迫切需要新的基于宿主机制的药物和治疗剂。由细胞内cAMP受体Epac 1和Epac 2介导的基于cAMP的细胞信号传导是cAMP效应转导的主要贡献者。我们已经报道了宿主Epac 1的基因缺失和药理学失活保护小鼠免受致命性立克次体病的侵害，并且在体外抑制Epac 1减少了立克次体对非吞噬细胞的粘附。其潜在机制仍不清楚。我们最近发现，立克次体劫持膜联蛋白A20-S100 A10（AnxA 2-p11）复合物介导的宿主纤溶机制，使其粘附于血管内皮细胞（EC）表面。此外，本发明还提供了一种方法， 我们的初步数据显示Epac 1基因的缺失或Epac 1的药物失活显著抑制了Anx A2和p11之间的结合，减少了EC外表面上的Anx 2-p11复合物，并减弱了内皮纤维蛋白溶解。本申请的中心假设是宿主Epac 1通过调节立克次体结合受体AnxA 2-p11复合物的形成来控制立克次体在流动血液的微环境中粘附到EC表面。正如我们的同行评审联合出版物和初步研究数据所证明的那样，我们在新方法学方面的成功合作和经验鼓励我们承担三个具体目标：目标1：验证Epac 1通过修饰AnxA 2调节EC中立克次体结合受体AnxA 2-p11复合物的形成来控制立克次体粘附的假设。目标二：检验Epac 1失活改变AnxA 2-p11复合物介导的EC顶端表面功能性地形特征，从而影响立克次体粘附的假设。目标3：验证宿主Epac 1的遗传和药理学失活可以减弱立克次体在体内血流动力学剪切力下粘附于血管腔表面的假设。我们的主要目标是描绘生化和生物力学机制的关键作用的主机Epac 1在致命的立克次体感染。由此产生的结果 拟议的研究将代表我们对立克次氏体感染的理解，以及对这些可怕的人类疾病的新药物和潜在治疗方法的发展的重大进展。

 DESCRIPTION (provided by applicant): Rickettsioses represent devastating human infections. Epidemic typhus and Rocky Mountain spotted fever (RMSF) are two of the most lethal infections known to humans. Although rickettsial infections can be controlled by appropriate broad-spectrum antibiotic therapy if diagnosed early, up to 20% of misdiagnosed or untreated and 5% of treated RMSF cases can be fatal. In addition, high infectivity and severe illness after inhalation make rickettsiae potential bioterrorism threats. However, a vaccine is not available for fatal rickettsioses, and novel host mechanism-based prophylactics and therapeutics are urgently needed. cAMP-based cell signaling mediated by intracellular cAMP receptors, Epac1 and 2, are major contributors to the transduction of the effects of cAMP. We have reported that both genetic depletion and pharmacological inactivation of the host Epac1 protected mice from fatal rickettsioses, and in vitro inhibition of Epac1 reduced rickettsial adherence to nonphagocytic cells. The underlying mechanisms remain unknown. We recently found that rickettsiae hijack the annexin A20-S100A10 (AnxA2-p11) complex-mediated host fibrinolytic machinery for their adherence to vascular endothelial cell (EC) surfaces. In addition, our preliminary data show that the deletion of the Epac1 gene or pharmacological inactivation of Epac1 significantly inhibits the associate between AnxA2 and p11, reduces the Anx2-p11 complex on external surfaces of ECs, and attenuates endothelial fibrinolysis. The central hypothesis of this application is that host Epac1 governs rickettsial adhesion to the EC surface in the microenvironment of flowing blood by regulating the formation of the rickettsial binding receptor AnxA2-p11 complex. Our successful collaboration and experience with novel methodologies, as demonstrated in our peer- reviewed joint publications and data from preliminary studies, encourage us to undertake three Specific Aims: Aim 1: To test the hypothesis that Epac1 governs rickettsial adhesion by regulating the formation of the rickettsial binding receptor AnxA2-p11 complex in EC through modification of AnxA2. Aim 2: To test the hypothesis that inactivation of Epac1 alters AnxA2-p11 complex-mediated functional topographical features of the EC apical surface, thereby affecting rickettsial adhesion. Aim 3: To test the hypothesis that genetic and pharmacological inactivation of host Epac1 can attenuate rickettsial adherence to the blood vessel luminal surface under hemodynamic shear stress in vivo. Our major goals are to delineate both biochemical and biomechanical mechanisms underlying the critical role of host Epac1 during a fatal rickettsial infection. Outcomes from this proposed research will represent a major advance in our understanding of rickettsial infection, and in the development of novel prophylactics and potential therapeutics for these dreadful human diseases.