PROJECT SUMMARY / ABSTRACT The Rickettsiae are a diverse group of Gram-negative, obligate intracellular bacterial pathogens that cause human diseases, including typhus and spotted fever. Among the causative agents of spotted fever group (SFG) rickettsiosis in the U.S., Rickettsia parkeri has proven to be a uniquely powerful model for studying pathogenicity at the cellular level, because it causes a non-lethal eschar-associated disease and therefore can be grown under biosafety level 2 conditions, facilitating cell biological studies. However, progress toward developing R. parkeri as a model for studying the innate immune response to SFG Rickettsia infection has been hindered by a dearth of studies employing mutant mice. As such, key questions regarding the innate immune response to SFG Rickettsia remain unanswered, including how the bacteria respond to type I interferon (IFN-I), an important cytokine of the innate immune system. Our new preliminary data indicate that intradermal infection of mice lacking both receptors for IFN-I and type II interferon (IFN-g) with R. parkeri results in a necrotic lesion at the site of infection and occasional lethality, whereas mice lacking each individual receptor exhibit no symptoms. This demonstrates a role for IFN-I (and IFN-g) in restricting R. parkeri growth in vivo. Moreover, the pathology of the double mutant is similar to (but more severe than) that occurring during human infection, suggesting it may represent a new animal model of human disease. We have also observed that IFN-I severely restricts R. parkeri growth in primary mouse macrophages, and that this is partially due to killing by the IFN-I regulated gene products, including antimicrobial guanylate binding proteins (GBPs). Despite these advances, there remain two key gaps in knowledge: (1) it is unclear how IFN-I restricts R. parkeri growth at the organ/tissue/cellular level in vivo; and (2) it is not known how gene products upregulated by IFN-I kill R. parkeri at the cellular/molecular level. We hypothesize that that IFN-I plays an important role in restricting Rickettsia growth in vivo and in vitro via the upregulation of cytosolic antibacterial molecules. We will test this hypothesis in two aims. In Aim 1, we will characterize the kinetics of intravenous and intradermal infection, the resulting organ and tissue pathologies, and the cell types infected by R. parkeri in mice lacking both receptors for IFN-I/IFN-g. These studies will reveal how IFN-I restricts growth of R. parkeri in vivo and will establish a robust murine model for investigating SFG Rickettsia pathogenesis. In Aim 2, we will test the effect of activating or mutating candidate antimicrobial factors (identified by RNAseq) on bacterial killing downstream of IFN-I signaling, and will test whether the GBPs restrict R. parkeri growth in endothelial cells in vitro and mice in vivo. Our findings will reveal how IFN-I restricts the growth of R. parkeri, and perhaps other microbes, in vit...