Epac1 Plays a Critical Role in Bacterial Adhesion during Rickettsioses

 描述(申请人提供)：立克次体病代表破坏性的人类感染。流行性斑疹伤寒和落基山斑点热(RMSF)是人类已知的两种最致命的感染。尽管如果及早诊断可以通过适当的广谱抗生素治疗来控制立克次体感染，但高达20%的误诊或未治疗以及5%的已治疗RMSF病例可能是致命的。此外，高传染性和吸入后的严重疾病使立克次体具有潜在的生物恐怖主义威胁。然而，疫苗并不是 可用于致死性立克次体病，迫切需要新的基于宿主机制的预防和治疗药物。细胞内cAMP受体Epac1和2介导的基于cAMP的细胞信号是cAMP效应转导的主要贡献者。我们已经报道，宿主Epac1的基因耗尽和药物灭活都能保护小鼠免受致死性立克次体感染，体外抑制Epac1可以减少立克次体与非吞噬细胞的黏附。其潜在机制仍不清楚。我们最近发现立克次体劫持了膜联蛋白A20-S100A10(AnxA2-p11)复合体介导的宿主纤溶机制，因为它们附着在血管内皮细胞(EC)表面。此外, 我们的初步数据显示，Epac1基因的缺失或Epac1的药物失活显著抑制AnxA2与p11之间的联系，减少内皮细胞外表面的Anx2-p11复合体，并减弱内皮纤溶。这一应用的中心假设是，宿主Epac1通过调节立克次体结合受体AnxA2-p11复合体的形成来调控立克次体与EC表面的黏附。我们在新方法方面的成功合作和经验，如我们同行评议的联合出版物和初步研究的数据所表明的那样，鼓励我们实现三个具体目标：目标1：检验Epac1通过修改AnxA2调控EC中立克次体结合受体AnxA2-p11复合体的形成来调控立克次体黏附的假说。目的：验证Epac1失活改变AnxA2-p11复合体介导的EC根尖表面功能形态特征，从而影响立克次体黏附的假说。目的：验证宿主Epac1基因失活和药物灭活可减轻血流动力学切应力作用下立克次体与血管管腔表面黏附的假说。我们的主要目标是描述宿主Epac1在致死性立克次体感染过程中的关键作用的生化和生物力学机制。由此产生的结果 拟议的研究将代表着我们在理解立克次体感染以及开发针对这些可怕的人类疾病的新的预防和潜在治疗方法方面的重大进展。

 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.