Effect Of SsaJ,ssaV,ssaG Gene On Salmonella Enteritidis Persistence In Macrophages

Salmonella spp.is an important zoonotic pathogen,its infection can seriously threaten animal and human health.Salmonella can persist in the host for a long time for immune escape,but this mechanism is not completely clear.Macrophage is the main carrier of systemic Salmonella infection,and Salmonella persistence in macrophage is the basis of that in the host.After invading macrophages,Salmonella can survive in SCV,which is main regulated by T3SS2.Needle Complex（NC）of Salmonella T3SS2 is a channel to transport effectors to cellular cytoplasm to play their function.Although there are a few studies on T3SS2 to mediate Salmonella persistence in macrophages and SCV integrity,NC is few attention.In previous study,Salmonella Enteritidis（SE）C50041ΔspiC was found to persist in macrophages and induce high level of intracellular IL-10.In this study,accroding to C50041ΔspiC,three representative genes ssaJ,ssaV and ssaG of the T3SS2 NC were focused on,to study mutants ability of proliferation.And analyze the relationship between the persistence of three mutants in macrophages and the integrity of SCV,it was further proved that T3SS2 NC could mediate Salmonella persistence in macrophages.1.Construction and biological features of ssaJ,ssa V and ssaG mutantsBased on the principle of homologous recombination,C50041ΔssaJ,C50041ΔssaV and C50041ΔssaG were constructed.Their biological features were further investigated.Compared with C50041,three mutants had no significant difference in the bacterial growth,biochemical characteristics,biofilm forming and motility;there was also no significant difference in the adhesion and invasion ability to RAW264.7.In macrophage,ssaJ,ssaV and ssaG three mutants can continually proliferate in macrophages for 42 hours,meanwhile high level of intracellular IL-10 was induced,which is consistent with C50041ΔspiC.By qPCR,mRNA levels of cellular pro-inflammatory-related cytokines IFN-γ,iNOS and SOCS3 were down-regulated and that of anti-inflammatory factors IL-10 and Arg-1 were significantly upregulated after RAW264.7 cells were infected by three mutants.The cytotoxicity of three mutants to macrophages was significantly lower than C50041 at 5 h and 8 h based on LDH assay,ssaJ,ssa V and ssaG three mutants decreases the exit ability of SE from macrophages.In vivo assay showed that LD₅₀ of ssaJ,ssaV and ssaG mutants increased over 1,500 folds compared with C50041,and they could not colonize in the spleen and liver,but IL-10 levels were elevated in the serum on the 14^th day post of mutants infection in mice.These results indicated that the virulence of ssaJ,ssaV and ssaG three mutants had significantly decreased.2.Effects of ssaJ,ssa V and ssaG mutants on SCV integrity in macrophagesIn macrophages,Salmonella can survive in SCV.The integrity of SCV is crucial for the intracellular persistence of Salmonella,and SifA is closely related to the integrity of SCV,and so the expression of effector protein SifA and the integrity of SCV were analyzed.The TEM-1 reporting system showed that ssaJ,ssaV,and ssaG mutant do not change the transport of effector protein SifA..The colocalization of three mutants with SCV could be observed by Confocal laser scanning microscopy（CLSM）.Colocalization rate between C50041 and SCV is 12.75%,but that between ssaJ,ssaV,and ssaG mutants and SCV is 36.70%,33.90%,26.73%,respectively.LLO can destroy SCV membrane,further LLO recombinant Salmonella was constructed.The proliferation ability of LLO recombinant Salmonella in macrophage was higher at 5 h but was lower at 20 h compared with ssaJ,ssa V,ssaG mutants.Colocalization of C50041ΔssaJ::LLO with SCV was significantly less than C50041 and C50041ΔssaJ,colocalization rate between C50041ΔssaJ::LLO and SCV is down to 5%.Based on the TEM-1 reporting system and WB,SpiC was found not to be secreted into the cytoplasm,but SpiC can be expressed in ssaJ,ssa V and ssaG three mutantsIn summary,Compared with C50041,ssaJ,ssa V and ssaG three mutants could persist in macrophages,induce high level of IL-10 in macrophages,and significantly reduce virulence in mice.They did not change SifA expression and translocation,and SCV integrity.But because of incomplete NC,they could not transfer SpiC.This study will broaden our understanding of T3SS2 Salmonella NC and bring new ideas for prevention and control to Salmonella.