Differential inflammation by Salmonella serovars is independent of FliC inflammasome activation

Abstract Salmonella Typhimurium and Salmonella Choleraesuis display distinct clinical manifestations, yet the molecular basis for their differential inflammatory potential, particularly via outer membrane vesicles (OMV), are not fully understood. Here, we examined the role of FliC in the differential inflammatory responses induced by S. Typhimurium UK-1 and S. Choleraesuis C78-3 and their corresponding OMV. In vivo, UK-1 infection triggered significantly elevated serum levels of IL-1β and IL-6 compared with C78-3 infection, a difference that was also observed following OMV administration. Isogenic fliC deletion mutants, which lacked both flagella and OMV-associated flagellin, were successfully generated. While wild-type bacteria and their OMV robustly activated the NLRC4 inflammasome pathway in bone marrow-derived macrophages (BMDMs), resulting in caspase-1 cleavage and IL-1β maturation, the Δ fliC mutants and their OMV exhibited markedly diminished activity. Notably, recombinant FliC proteins from the two serovars, despite sharing only 77% amino acid homology, activated the inflammasome with comparable efficiency. A key finding was that UK-1(Δ fliC ) and its OMV still induced higher IL-1β production than C78-3(Δ fliC ) counterparts both in vitro and in vivo, indicating that the enhanced immunostimulatory capacity of UK-1 is mediated by factors independent of FliC. Collectively, this study provides new insights into the serovar-specific inflammatory mechanisms of Salmonella and suggests that FliC-independent factors warrant consideration in the development of OMV-based vaccines and the understanding of inflammatory responses.