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Salmonella enterica serovar Typhimurium is an intracellular pathogen that parasitizes macrophages from within a vacuole. The vacuolar environment prompts the bacterium to regulate the lipid composition of the outer membrane (OM), and this influences host inflammation. S. Typhimurium regulates the levels of acidic glycerophospholipids known as cardiolipins (CL) within the OM, and mitochondrial CL molecules can prime and activate host inflammasomes. However, the contribution of S. Typhimurium's CL biosynthesis genes to intracellular survival, inflammasome activation, and pathogenesis had not been examined. S. Typhimurium genes encode three CL synthases. Single, double, and triple mutants were constructed. Similar to other Enterobacteriaceae, ClsA is the primary CL synthase for S. Typhimurium during logarithmic growth, while ClsB and ClsC contribute CL production in stationary phase. It was necessary to delete all three genes to diminish the CL content of the envelope. Despite being devoid of CL molecules, ΔclsABC mutants were highly virulent during oral and systemic infection for C57BL/6J mice. In macrophages, ΔclsA, ΔclsB, ΔclsC, and ΔclsAC mutants behaved like the wild type, whereas ΔclsAB, ΔclsBC, and ΔclsABC mutants were attenuated and elicited reduced amounts of secreted interleukin-1 beta (IL-1β), IL-18, and lactate dehydrogenase. Hence, when clsA and clsC are deleted, clsB is necessary and sufficient to promote intracellular survival and inflammasome activation. Similarly, when clsB is deleted, clsA and clsC are necessary and sufficient. Therefore, the three CL synthase genes cooperatively and redundantly influence S. Typhimurium inflammasome activation and intracellular survival in C57BL/6J mouse macrophages but are dispensable for virulence in mice. IMPORTANCE Salmonella enterica serovar Typhimurium is a pathogenic Gram-negative bacterium that regulates the cardiolipin (CL) and lipopolysaccharide (LPS) composition of the outer membrane (OM) during infection. Mitochondrial CL molecules activate the inflammasome and its effector caspase-1, which initiates an inflammatory process called pyroptosis. Purified bacterial CL molecules also influence LPS activation of Toll-like receptor 4 (Tlr4). S. Typhimurium resides within macrophage vacuoles and activates Tlr4 and the inflammasome during infection. However, the contribution of the three bacterial CL synthase genes (cls) to microbial pathogenesis and inflammation had not been tested. This study supports that the genes encoding the CL synthases work coordinately to promote intracellular survival in macrophages and to activate the inflammasome but do not influence inflammatory cytokine production downstream of Tlr4 or virulence in C57BL/6J mice. The macrophage phenotypes are not directly attributable to CL production but are caused by deleting specific combinations of cls gene products.