Host GPCR-cAMP signaling balances Gαs and Gαi activity to control intracellular <i>Brucella</i> infection

In this study, we investigated the impact of G protein-coupled receptor (GPCR) signaling on the intracellular replication of the model pathogen <i>Brucella neotomae</i>. Building on a prior chemical genetics screen, we identified agonists of the Gαi-coupled adenosine A1 and dopamine D4 receptors as potent inhibitors of intracellular Brucella replication. In contrast, agonists of Gαs-coupled adenosine A2A or dopamine D1 receptors, as well as antagonists of A1 or D4 receptors, either failed to inhibit or enhanced intracellular replication. Wild-type <i>B. neotomae</i> induced a rapid, type IV secretion system-dependent increase in host-cell cAMP during early infection. ENBA and cilostamide prevented this infection-associated cAMP increase and completely inhibited intracellular growth; this effect was partially reversed by cell-permeable cAMP analogs Using a real-time NanoBRET biosensor, we detected rapid Gαs activation within minutes of infection that was sustained during wild-type but not ΔvirB4 infection and was abrogated by ENBA or cilostamide. Disruption of early Gαs-cAMP signaling redirected BCVs to replication-incompatible phagolysosomal and autophagy-associated compartments. Collectively, these data support a model in which early GPCR signaling dynamics, balancing Gαs and Gαi pathways, are critical for establishment of productive intracellular Brucella infection.