Salmonella Enteritidis antitoxin DinJ inhibits NLRP3-dependent canonical inflammasome activation in macrophages

The inflammasome is a pivotal component of the innate immune system, acting as a multiprotein complex that plays an essential role in detecting and responding to microbial infections. Salmonella Enteritidis have evolved multiple mechanisms to regulate inflammasome activation and evade host immune system clearance. Through screening S. Enteritidis C50336 Delta fliC transposon mutant library, we found that the insertion mutant of dinJ increased inflammasome activation. In this study, we demon strated the genetic connection between the antitoxin DinJ and the toxin YafQ in S. Enteritidis, confirming their co -transcription. The deletion mutant Delta fliC Delta dinJ increased cell death and IL-1 beta secretion in J774A.1 cells. Western blotting analysis further showed elevated cleaved Caspase-1 product (p10 subunits) and IL-1 beta secretion in cells infected with Delta fliC Delta dinJ compared to cells infected with Delta fliC. DinJ was found to inhibit canonical inflammasome activation using primary bone marrow -derived macrophages (BMDMs) from Casp-/- C57BL/6 mice. Furthermore, DinJ specifically inhibited NLRP3 inflammasome activation, as demonstrated in BMDMs from Nlrp3(-/- )and Nlrc4(-/-) mice. Fluorescence resonance energy transfer (FRET) experiments confirmed the translocation of DinJ into host cells during infection. Finally, we revealed that DinJ could inhibit the secretion of IL-1 beta and IL -18 in vivo , contributing to S. Enteritidis evading host immune clearance. In summary, our findings provide insights into the role of DinJ in modulating the inflam masome response during S. Enteritidis infection, highlighting its impact on inhibiting inflammasome activation and immune evasion.