Pp6-Pfkfb1 axis modulates intracellular bacterial proliferation by orchestrating host-pathogen metabolic crosstalk

Abstract

Intracellular bacterial pathogens exhibit heterogeneous replication rates within host macrophages, but the mechanisms by which they manipulate host factors for survival remain incompletely understood. Using a fluorescence-dilution reporter system in Salmonella typhimurium (Salmonella)-infected macrophages, we found that Protein Phosphatase 6 (Pp6) was downregulated in macrophages harboring growing bacteria. Conditional knockout of Pp6 elevated host susceptibility to Salmonella-mediated lethality due to compromised antimicrobial defenses. MicroRNA-31 (miR-31) was identified as a negative regulator of Pp6, and its conditional ablation enhanced bacterial clearance. Yeast two-hybrid screening identified 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 (Pfkfb1), a metabolic regulator, as a substrate of Pp6. Pp6 deficiency resulted in significantly elevated expression of Pfkfb1, which was highly expressed in macrophages containing replicating Salmonella. Pfkfb1 deletion restricted bacterial proliferation by promoting nitric oxide (NO) production while concurrently suppressing arginase-1 (Arg-1) expression and impairing arginine metabolism in macrophages. Collectively, these results establish the Pp6-Pfkfb1 axis as a key regulator of host metabolic adaptation and intracellular bacterial survival, offering potential therapeutic targets against multidrug-resistant pathogens. Author summaryIntracellular pathogens secrete effector proteins that intercept and modify host cells to usurp host defenses and establish habitable intracellular niches, yet the host factors they manipulate for survival and virulence are not fully understood. Using a fluorescence-dilution reporter system in Salmonella typhimurium (Salmonella)-infected macrophages, we found Protein Phosphatase 6 (Pp6) was significantly decreased in macrophages harboring replicating Salmonella and its deficiency indulged bacterial growth in macrophages. However, the deletion of microRNA-31 (miR-31), a negative regulator of Pp6, enhanced Salmonella clearance. Furthermore, through yeast two- hybrid screening, we identified 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 (Pfkfb1) is a substrate of Pp6. And we demonstrated Pfkfb1, abundantly expressed in macrophages containing growing bacteria, is crucial for Salmonella proliferation by regulating nitric oxide (NO) levels, arginase-1 (Arg-1) expression and arginine metabolism. Overall, the molecular changes in Pp6-Pfkfb1 axis drive host metabolic adaptations that enable intracellular bacterial survival.