PROJECT SUMMARY Rickettsia species are arthropod-borne obligate intracellular bacteria with both symbiotic and pathogenic lifecycles. The global impact of rickettsial infections is highlighted by the resurgence of human infections with R. rickettsii (etiologic agent of Rocky Mountain Spotted Fever) in Central and South America, reappearance of R. conorii (etiologic agent of Boutonneuse Fever) in Europe, Middle East, and Africa9 and the recent outbreaks of R. rickettsii and R. typhi (etiologic agent of murine typhus) in the USA. Unfortunately, our inadequate understanding of Rickettsia-host interaction, in particular at the level on rickettsial engulfment via receptor-ligand- mediated phagocytosis on professional host defense cells, like macrophages (MΦ), has significantly impaired the development of effective therapeutics against these pathogens. To address these knowledge gaps, we propose to: i) characterize the specific rickettsial outer membrane glycerophospholipid ligand(s) involved in the phagocytic process of SFG and TG rickettsiae by MΦ, ii) decipher the role of efferocytic receptor, CD300f, in facilitating host colonization of virulent Rickettsia species. Our preliminary work identified phosphatidylserine (PS), a well-characterized “eat-me” signal involved in the phagocytosis of apoptotic cells (aka efferocytosis), as a putative ligand on outer membrane of rickettsiae. We further validated PS as crucial ligand for engulfment of R. typhi and R. rickettsii [Shelia Smith], two virulent bacteria representing the TG and SFG, respectively, by using recombinant Annexin V, a molecule known to block PS-mediated efferocytosis, in bone marrow-derived macrophages (BMMΦ). As PS-dependent phagocytosis requires PS-receptor recognition, we focused on CD300f, a type I transmembrane cell surface receptors with a high binding affinity to PS. Using WT and CD300f- /- BMMΦ, we showed that engulfment of R. typhi and R. rickettsii was dependent on CD300f expression, which was further confirmed by αCD300f antibody-mediated neutralization assays on WT BMMΦ. We tested the role of CD300f in vivo, by establishing mouse models of rickettsiosis with ~LD50 for both R. typhi and R. rickettsii species in C57BL/6J WT mice and showed that, unlike WT mice, CD300f-/- animals were protected against Rickettsia-induced lethality. Furthermore, in vivo depletion of MΦ, via clodronate-liposomes, suggest that CD300f-expression on MΦ contributed to the protection against Rickettsia-induced lethality. Hence, we will test the hypothesis, that both SFG and TGs Rickettsia exploit efferocytic signaling to invade MΦ, using the following Aims: to identify rickettsial outer membrane glycerophospholipid [phosphatidylserine (PS) or other] ligand(s) involved in the engulfment and colonization of pathogenic Rickettsia by MΦ (Aim 1) and define the contributing role of glycerophospholipid-binding receptor, CD300f, in promoting engulfment and colonization of the host by pathogenic Rickettsia in MΦ...