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.