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

项目摘要/摘要 立克次体代表着毁灭性的人类感染。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。尽管立克次体感染是可以控制的 通过适当的广谱抗生素治疗，如果及早诊断，高达20%的误诊或未治疗 接受治疗的RMSF病例中有5%可能是致命的。此外，吸入后传染性高和病情严重 使立克次体成为潜在的生物恐怖主义威胁。播散性内皮细胞感染与内皮屏障 破裂伴微血管通透性增加是其主要的病理生理特征。然而，a 致死性立克次体没有疫苗，新的基于宿主机制的预防和治疗方法 都是迫切需要的。我们已经报道了环磷酸腺苷(CAMP)信号在其中起关键作用。 CAMP直接激活的cAMP交换蛋白(EPAC)信号在致死性立克次体发病中的作用 轴心。然而，上游信号通路在这一病理过程中的作用尚不完全清楚。腺苷 A2A型受体(A2AR)主要表达于血管内皮细胞。A2AR一直很好 被认为是cAMP信号的上游调节器，并能够刺激细胞内 营地积累。我们发现，批准的帕金森氏病(PD)药物KW6002，一种A2AR 拮抗剂在细胞模型和血管模型中均显著抑制立克次体感染。 KW6002灭活A2AR可削弱立克次体粘附素rOmpB对活体的纳米结合力 内皮细胞(ECs)，从而限制立克次体与ECs的黏附。这些发现表明，A2AR- CAMP信号通路可能通过作用于宿主立克次体结合受体S来控制立克次体感染 ECS。我们提出了我们的中心假设，即选择性A2AR拮抗剂KW6002可以提供保护 通过阻止细菌与内皮细胞的黏附来对抗立克次体感染的效果。为了检验这一假设，我们将 追求以下具体目标：目标1：确定A2AR在 立克次体粘连。目的2：明确立克次体与内皮细胞黏附的纳米力学机制 受A2AR调节的表面。目的3：评价KW6002是否能预防致死性立克次体 在体内通过阻止立克次体与内皮细胞衬里的黏附而感染。我们将通过以下方式验证我们的假设 使用尖端方法，即1)立克次体粘附素之间相互作用的功能成像 和活的EC表面，以及2)一种新的、基于解剖学的活体细菌黏附 测量系统。这些结果将为生物力学和分子机制提供更深入的见解。 但更重要的是，预计数据将支持科学证明 改变批准药物的用途，立即用于临床试验，与可怕的人类疾病作斗争 由立克次体引起。

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.