Host GPCR-cAMP signaling balances Gαs and Gαi activity to control intracellular Brucella infection

In this study, we investigated the impact of G protein-coupled receptor (GPCR) signaling on the intracellular replication of the model pathogen Brucella neotomae. Building on a prior chemical genetics screen, we identified agonists of the Gi-coupled adenosine A1 and dopamine D4 receptors as potent inhibitors of intracellular Brucella replication. In contrast, agonists of Gs-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 B. neotomae 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 Gs activation within minutes of infection that was sustained during wild-type but not {Delta}virB4 infection and was abrogated by ENBA or cilostamide. Disruption of early Gs-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 Gs and Gi pathways, are critical for establishment of productive intracellular Brucella infection. Importance StatementBrucella species cause chronic infections by surviving and multiplying inside immune cells. To do this, Brucella must remodel the membrane-bound compartment that surrounds it after uptake, steering it away from destructive lysosomes and toward a permissive niche where replication can occur. We found that Brucella rapidly triggers a host signaling response controlled by G protein-coupled receptors, leading to a rise in a common cellular messenger molecule (cAMP) within minutes of infection. This early signal depends on the bacterial type IV secretion system and is required to build the replication-permissive compartment. When we disrupted this signaling with small molecules, bacteria were rerouted into degradative, autophagy-associated compartments and failed to establish productive infection. These results reveal an early host signaling checkpoint that Brucella exploits to create its intracellular niche and suggest that targeting host signaling dynamics, rather than bacterial viability directly, may provide new strategies to block intracellular infection.