Francisella tularensis Catalase Restricts Immune Function by Impairing TRPM2 Channel Activity*

As an innate defense mechanism, macrophages produce reactive oxygen species that weaken pathogens and serve as secondary messengers involved in immune function. The Gram-negative bacterium Francisella tularensis utilizes its antioxidant armature to limit the host immune response, but the mechanism behind this suppression is not defined. Here we establish that F. tularensis limits Ca²⁺ entry in macrophages, thereby limiting actin reorganization and IL-6 production in a redox-dependent fashion. Wild type (live vaccine strain) or catalase-deficient F. tularensis (ΔkatG) show distinct profiles in their H₂O₂ scavenging rates, 1 and 0.015 pm/s, respectively. Murine alveolar macrophages infected with ΔkatG display abnormally high basal intracellular Ca²⁺ concentration that did not increase further in response to H₂O₂. Additionally, ΔkatG-infected macrophages displayed limited Ca²⁺ influx in response to ionomycin, as a result of ionophore H₂O₂ sensitivity. Exogenously added H₂O₂ or H₂O₂ generated by ΔkatG likely oxidizes ionomycin and alters its ability to transport Ca²⁺. Basal increases in cytosolic Ca²⁺ and insensitivity to H₂O₂-mediated Ca²⁺ entry in ΔkatG-infected cells are reversed by the Ca²⁺ channel inhibitors 2-aminoethyl diphenylborinate and SKF-96365. 2-Aminoethyl diphenylborinate but not SKF-96365 abrogated ΔkatG-dependent increases in macrophage actin remodeling and IL-6 secretion, suggesting a role for H₂O₂-mediated Ca²⁺ entry through the transient receptor potential melastatin 2 (TRPM2) channel in macrophages. Indeed, increases in basal Ca²⁺, actin polymerization, and IL-6 production are reversed in TRPM2-null macrophages infected with ΔkatG. Together, our findings provide compelling evidence that F. tularensis catalase restricts reactive oxygen species to temper macrophage TRPM2-mediated Ca²⁺ signaling and limit host immune function.