Targeting the Host Adenosine A2A Receptor to Protect Against Fatal Rickettsiosis Using an Approved Parkinson's Disease Drug

项目总结/摘要 立克次体病是毁灭性的人类传染病。流行性斑疹伤寒与落基山斑疹热 （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控制 如果早期诊断，通过适当的广谱抗生素治疗，高达20%的误诊或未治疗 并且5%的治疗的RMSF病例可能是致命的。此外，吸入后传染性高，病情严重 使立克次体成为潜在的生物恐怖主义威胁。播散性内皮感染与内皮屏障 微血管通透性增加的破坏是主要的病理生理学特征。但 致命性立克次体病尚无疫苗，基于宿主机制的新型药物和治疗剂 是迫切需要的。我们已经报道了环磷酸腺苷（cAMP）信号转导在细胞凋亡中起着关键的作用。 在致死性立克次体病中通过cAMP交换蛋白（Epac）信号直接激活的作用 轴线然而，上游信号通路在这种病理中的作用是完全未知的。腺苷 A2 A受体（A2 AR）主要在血管内皮细胞（EC）中表达。A2 AR一直很好。 被认为是cAMP信号传导的上游调节因子，能够刺激细胞内的 cAMP积累。我们发现，批准的帕金森病（PD）药物KW 6002，一种A2 AR， 拮抗剂，在细胞模型和脉管系统模型中都显著抑制立克次体感染。 KW 6002灭活A2 AR可减弱立克次体粘附素rOmpB对活体的纳米结合力 内皮细胞（EC），从而限制立克次体对EC的粘附。这些发现表明，A2 AR- cAMP信号通路可能通过作用于宿主立克次体结合受体来控制立克次体感染， EC。我们提出了我们的中心假设，即选择性A2 AR拮抗剂KW 6002可以提供保护性 通过阻止细菌粘附于内皮细胞来对抗立克次体感染的功效。为了验证这个假设，我们将 目的1：鉴定A2 AR在血管内皮细胞增殖过程中调节的内皮细胞表面靶点。 立克次体粘附目的2：探讨立克次体粘附内皮细胞的纳米力学机制 由A2 AR监管的表面。目的3：评价KW 6002对致死性立克次体的保护作用 通过在体内阻止立克次体粘附于内皮衬里来预防感染。我们将测试我们的假设， 采用尖端的方法，即，1)立克次体粘附素相互作用的功能成像 和活EC表面使用AFM，和2）一种新的，基于解剖学的，在体内定量细菌粘附 测量系统结果将提供更深入的了解生物力学和分子机制 立克次体感染，但更重要的是，预计数据将支持科学的理由 将一种已获批准的药物重新用于临床试验，以对抗可怕的人类疾病， 由立克次体引起的

PROJECT SUMMARY/ ABSTRACT 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. Disseminated endothelial infection and endothelial barrier disruption with increased microvascular permeability are the central pathophysiologic features. However, a vaccine is not available for fatal rickettsioses, and novel host mechanism-based prophylactics and therapeutics are urgently needed. We have reported that cyclic adenosine monophosphate (cAMP) signaling plays a critical role during fatal rickettsioses through the cAMP-exchange protein directly activated by cAMP (Epac) signaling axis. However, the role of upstream signaling pathway in this pathology is completely unknown. Adenosine A2A receptor (A2AR) is predominantly expressed in vascular endothelial cells (ECs). A2AR has been well recognized to serve as an upstream regulator of cAMP signaling and to be able to stimulate the intracellular cAMP accumulation. We found that the approved Parkinson’s disease (PD) drug KW6002, an A2AR antagonist, dramatically suppressed rickettsial infection in both a cell model and a vasculature model. Inactivation of A2AR by KW6002 weakens the nano-binding force of the rickettsial adhesin rOmpB to living endothelial cells (ECs), thereby limiting rickettsial adherence to ECs. These findings suggest that the A2AR- cAMP signaling pathway may control rickettsial infection by acting on host rickettsial-binding receptor(s) in ECs. We propose our central hypothesis that the selective A2AR antagonist KW6002 can provide protective efficacy against rickettsial infection by impeding bacterial adhesion to ECs. To test this hypothesis, we will pursue the following Specific Aims: Aim 1: To identify endothelial surface targets regulated by A2AR during rickettsial adhesion. Aim 2: To define the nanomechanical mechanism of rickettsial adhesion to endothelial surfaces regulated by A2AR. Aim 3: To evaluate whether KW6002 can protect against lethal rickettsial infection by arresting rickettsial adherence to the endothelial lining in vivo. We will test our hypothesis by employing cutting-edge approaches, i.e., 1) functional imaging of the interactions between rickettsial adhesins and living EC surfaces using AFM, and 2) a novel, anatomy-based, in vivo quantitative bacterial adhesion measuring system. Outcomes will provide deeper insights into the biomechanical and molecular mechanisms of rickettsial infections, but more importantly it is expected that the data will support the scientific justification for repurposing an approved drug for immediate use in clinical trials to battle the dreadful human diseases caused by Rickettsia.