Salmonella Enteritidis SpaO protein regulating inflammasome activation in macrophage.

Abstract

Activation of the inflammasome plays a critical role in host defense against Salmonella infection, yet the bacterial factors governing this process remain poorly defined. Thus, we constructed a transposon mutant library of Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis C50336ΔfliC) to identify the genes involved in inflammasome activation in the macrophage J774A.1. Out of 2639 mutants, seven strains exhibited influence the inflammasome activation, and spaO was selected for further investigation. Compared with the ΔfliC infection group, infection with the ΔfliCΔspaO strain significantly reduced lactate dehydrogenase (LDH) release and secretion of IL-1β and IL-18. Cleaved Caspase-1 and GSDMD were also decreased in macrophage infection with ΔfliCΔspaO, indicating that SpaO is required for inflammasome activation. Consistent results were confirmed in bone marrow-derived macrophages (BMDMs), and we further found that SpaO is required for activation of both NLRC4 and NLRP3 inflammasomes. In a mouse oral infection model, ΔfliCΔspaO infected mice exhibited significantly reduced weight loss, mortality, and bacterial loads in spleen and liver compared with the ΔfliC group. Correspondingly, serum levels of IL-1β and IL-18 were markedly decreased, further supporting the role of SpaO in promoting inflammasome activation in vivo. Moreover, we found that SpaO regulates inflammasome activation maybe by influencing the secretion of the T3SS1 effectors SipB and SipC. Collectively, these findings reveal that SpaO is an important T3SS1 component required for efficient inflammasome activation and virulence of S. Enteritidis, advancing our understanding of bacterial modulation of host innate immunity.