Project Summary/Abstract In the United States, Ixodes scapularis ticks harbor and transmit several pathogens including human anaplasmosis bacterial agent Anaplasma phagocytophilum. This bacterium is transmitted to the vertebrate host by an infected tick bite. Several studies have addressed molecular mechanisms that A. phagocytophilum uses to survive in the mammalian host. Relatively, few studies have clearly defined the molecular strategies that this bacterium uses to survive in ticks. In the previous funding period, we performed a comprehensive molecular analysis on I. scapularis organic anion transporting polypeptides 4056 (IsOATP4056) and genes involved in the tryptophan metabolism pathway in A. phagocytophilum-tick interactions. We made substantial progress and have published several research articles showing importance of IsOATP4056 and the tryptophan pathway in the survival and transmission of A. phagocytophilum from ticks to the naïve vertebrate host. Passive Immunization with IsOATP4056 antibodies impaired A. phagocytophilum transmission from ticks to the vertebrate host. Anaplasma phagocytophilum upregulates IsOATP4056 and activates the tryptophan pathway leading to the increased synthesis of endogenous levels of tryptophan metabolite, Xanthurenic acid (XA). The increased XA prevents the build-up of reactive oxygen species facilitating bacterial survival in tick cells. In addition, A. phagocytophilum activates the XA-mediated p38-MAPK pathway to inhibit tick cell death thereby facilitating the survival of both the bacterial agent and its vector host. We also reported a novel role for XA in the transcriptional activation of isoatp4056 gene expression. Furthermore, A. phagocytophilum downregulates microRNA (miR133) that targets isoatp4056 for its survival and transmission from vector to the vertebrate host. These findings from our previous funding period provide a strong rationale for the proposed aims for this R01 (competing renewal) application. In Aim 1, we propose to understand the mechanism of how passive immunization affects the transmission of A. phagocytophilum from ticks to the vertebrate host. In Aim 2, we will analyze whether targeting IsOATP4056 via active immunization or OATP inhibitor treatment affects the transmission of A. phagocytophilum from ticks to the vertebrate host. In Aim 3, we will characterize XA-mediated regulation of miRNAs that are important in regulating gene expression during A. phagocytophilum survival and transmission from infected ticks to the naïve host. Based on the success in the previous funding period and the aims proposed in this renewal application, we believe that this could be a transformative study that not only serves as a model to study intimate relationships established by pathogens with their arthropod vectors but may also lead in the development of new strategies to interrupt the transmission of this and perhaps other rickettsial bacteria of medical importance.