An arthropod transporter IsOATP4056 exerts inhibitory effect on its interacting membrane protein to facilitate rickettsial pathogen survival in ticks

The black-legged tick, Ixodes scapularis, transmits several medically important pathogens to humans including Anaplasma phagocytophilum. Our previous studies provided evidence that this bacterium modulates arthropod organic anion transporting polypeptide (IsOATP4056) and tryptophan pathways for its survival and transmission from ticks. In this study, we identified that IsOATP4056 interacts with I. scapularis hypothetical protein (IsHP) to suppress arthropod innate immunity thereby facilitating A. phagocytophilum survival in ticks and tick cells. Co-precipitation with recombinant IsHP and tick protein lysates followed by immunoblotting analysis with anti- IsOATP4056 antibody was performed to reveal whether these two proteins directly interact. RNAi-mediated silencing experiments were performed to understand the roles of IsHP, IsOATP4056 and Aryl hydrocarbon Receptor (AhR) in tick-A. phagocytophilum interactions. Immunoprecipitation and immunoblotting analysis revealed interaction of IsHP with IsOATP4056. RNAi-mediated silencing of ishp expression affected arthropod innate immune response that resulted in significantly increased bacterial burden in tick cells. In contrast, RNAi-mediated silencing of isoatp4056 expression or antibody-mediated blocking of IsOATP4056 upregulated ishp and innate immune response that controlled bacterial burden in ticks and tick cells. Furthermore, we noted that A. phagocytophilum infection or treatment with tryptophan metabolite xanthurenic acid (XA) significantly upregulates Aryl hydrocarbon Receptor (AhR) expression in ticks and tick cells. RNAi-mediated silencing of ahr expression decreased isoatp4056 transcripts and bacterial burden but increased the ishp expression. EMSA results further support that AhR and XA positively regulate isoatp4056 promoter. These results elucidate that A. phagocytophilum infection triggers AhR-mediated regulation of isoatp4056 expression to inactivate IsHP-associated pelle expression for its survival in ticks and tick cells.