| ObjectiveSalmonella enterica serotype Typhi(S.Typhi)Vi capsular polysaccharide is a unique virulence factor of S.Typhi.In macrophages,autophagy plays an important role in the elimination of intracellular Salmonella.But Salmonella also derives strategies to resist or utilize autophagy.Our research group previously used RNA-seq to analyze the transcriptome of S.Typhi infected macrophages and suggested that multiple autophagy-related genes were differentially expressed.In this study,we focus on the relationship between S.TyphiVi capsular polysaccharide and autophagy in macrophages to clarify the expression characteristics,function and mechanism of Vi capsular polysaccharide.Methods1.Analysis of autophagy level of S.Typhi infected macrophagesTotal RNA and total protein of S.Typhi infected macrophages in each phase were obtained.q RT-PCR was used to detect the m RNA levels of autophagy-related genes;Western blot was used to detect OPTN,p62,NDP52 and LC3.2.Analysis of the expression characteristics of S.TyphiVi capsular polysaccharide:(1)Extracellular: Total RNA of S.Typhi statically cultured in SOB and LB liquid medium for 2 h,8 h and 12 h were respectively acquired and the m RNA levels of tviA and tvi B were detected by q RT-PCR.(2)Intracellular: Detecting tviA and tvi B m RNA levels by q RT-PCR under different S.Typhi infection phases in macrophages;Detecting the fluorescence intensity of Vi capsular polysaccharide in macrophages under different infection phases by flow cytometry and immunofluorescence.3.Strain constructions(1)ΔtviA and Δvex E: the mutant strains of the S.TyphiVi capsular polysaccharide encoding genes tviA(regulation)and vex E(export)were prepared respectively by homologous recombination of suicide plasmid.(2)ΔtviA-p BAD33-tviA(C-ΔtviA)and Δvex E-p BAD33-vex E(C-Δvex E): The coding sequences of the tviA gene and vex E gene were respectively cloned into the p BAD33 expression plasmid and electrotransformed into ΔtviA and Δvex E to prepare ΔtviA complement strain(C-ΔtviA)and Δvex E complement strain(C-Δvex E).Meanwhile,The empty plasmid p BAD33 was introduced into the wild strains WT,ΔtviA and Δvex E as control strains(WT-p BAD33,ΔtviA-p BAD33,Δvex E-p BAD33).4.Analysis of the function of S.TyphiVi capsular polysaccharide gene tviA,vex E(1)The growth curve: In SOB liquid medium,growth curve of WT,ΔtviA andΔvex E under static culture conditions were drawn.The effect of Vi capsular polysaccharide coding genes on bacterial growth was analyzed.(2)The macrophages intracellular survival assay: C-ΔtviA,C-Δvex E,WT-p BAD33,ΔtviA-p BAD33 and Δvex E-p BAD33 were statically cultured in SOB medium and the expression was induced by L-arabinose.After co-culturing the bacteria and macrophages and killing the extracellular bacteria,cells were lysed and incubated on LB plate to count the colonies(T0).Other cells were incubated for a further 2,12 or 24 h and incubated on LB plates and counted colonies.The value of T2/T0,T12/T0 and T24/T0 were used to compare the bacteria intracellular survival ability.(3)The tviA and vex E effects on the autophagy of macrophages: C-ΔtviA,C-Δvex E,WT-p BAD33,ΔtviA-p BAD33 and Δvex E-p BAD33 were statically cultured in SOB medium and the expression was induced by L-arabinose.Collecting total protein of strains infecting macrophages and analyzing LC3 and p62 protein levels by western blot;Macrophages were infected with WT,ΔtviA,and Δvex E.The intracellular LC3 punctate aggregation was detected by immunofluorescence.The fluorescence intensity of intracellular LC3 was detected by flow cytometry.The m RNA levels of Nod1,Nod2 and Galectin8 genes were detected by q RT-PCR.Results1.Analysis of autophagy level of S.Typhi infected macrophages(1)Transcriptome data analysis of S.Typhi infected macrophages showed that the m RNA levels of at least 21 autophagy-related genes were differentially expressed during the 2 h and 24 h infection phases.q RT-PCR verified that the m RNA level changes of Rnf166,Atg4 c,Atg16l1,Rubcnl,Lamp3,Casp1,Dapk1 were generally consistent with the trends of transcriptome genes.(2)Dynamic analysis of S.Typhi infection and autophagy showed that compared with the uninfected group,the m RNA levels of Rnf166,Atg4 c,Atg16l1,Rubcnl,Lamp3,Casp1,Dapk1 increased within 30 minutes of infection.Most of the m RNAs of Ndp52,p62,Optn,Galectin8,Lamp1,Rubcn and Lc3 b were up-regulated within 6 hours of infection.However,m RNA of p62,Lc3 b and Lamp1 decreased slightly at 6h;Western blot results showed that OPTN,p62 proteins decreased and LC3-II increased after 2 h-6 h infection.In 12 h-24 h infection phase,OPTN and p62 protein expression increased significantly.NDP52 had not significant change.The content of LC3-II reached the highest in 24 h.2.Analysis of the expression characteristics of S.TyphiVi capsular polysaccharide(1)The expression analysis of S.TyphiVi capsular polysaccharide under different culture conditions showed that the m RNA level of S.Typhi tviA and tvi B in SOB liquid medium was higher than that of LB liquid medium.(2)Dynamic expression analysis of S.TyphiVi capsular polysaccharide in macrophages.q RT-PCR showed that the m RNA of tviA and tvi B could be detected within 30 min-24 h of infection and significantly decreased after 2 h;flow cytometry detected the fluorescence signal of Vi capsular polysaccharide in cells infected with S.Typhi from 2h-24 h.But the fluorescence intensity had no significant difference;the intracellular fluorescence signal of Vi capsular polysaccharide could be observed in each infection time detected by immunofluorescence and the fluorescence intensity was decreased after 2 h of infection.3.Strain constructions(1)S.Typhi ΔtviA,Δvex E strains were successfully constructed.(2)Successfully constructed S.Typhi ΔtviA-p BAD33-tviA(C-ΔtviA),Δvex E-p B AD33-vex E(C-Δvex E),WT-p BAD33,ΔtviA-p BAD33,Δvex E-p BAD33.4.Analysis of the function of S.TyphiVi capsular polysaccharide gene tviA,vex E(1)The growth curve showed that the growth rate of ΔtviA is significantly lower than that of WT.There was no significant difference between the growth curves of Δvex E and WT.(2)The macrophages intracellular survival assay showed that ΔtviA-p BAD33 andΔvex E-p BAD33 had significantly reduced survival ability in macrophages comparing with WT-p BAD33.(3)The analysis of S.TyphiVi capsular polysaccharide and the macrophages autophagy.Western blot results showed that compared with WT,most p62 protein and LC3-II had no significant difference at 2 h and 24 h after infection.In Δvex E infection group,LC3-II at 2 h and p62 protein at 24 h were slightly increased.At 1 h after infection,the p62 of the ΔtviA-p BAD33 andΔvex E-p BAD33 groups was decreased and the level of LC3-II was increased comparing with WT-p BAD33;the immunofluorescence and flow cytometry showed that the intracellular LC3 punctate aggregation and mean fluorescence intensity in the ΔtviA and Δvex E infection groups were significantly increased at 1 h.(4)Preliminary analysis of the molecular mechanism of S.TyphiVi capsular polysaccharide and macrophage autophagy.At 30 min and 1 h of infection,there was no significant difference in the m RNA of Nod1 and Nod2 in theΔtviA and Δvex E infection groups compared with the WT infection group.But Nod2 m RNA increased in the Δvex E infection group at 30 min.The level m RNA of Galectin8 increased for 1 h and decreased after 30 min of infection in the two infection groups.Conclusions1.S.Typhi can dynamically affect the transcription level of autophagy-related gene and protein expression in macrophages.S.Typhi can promote autophagy in the early infection stage and may inhibited autophagy in the late infection stage.2.In SOB medium,Vi capsular polysaccharide was expressed at a high level.Its coding gene tviA promoted bacterial growth while vex E had no obvious effect.3.In macrophages,Vi capsular polysaccharide were continuous expression.Both tviA and vex E promoted the intracellular survival of S.Typhi and impaired autophagy in macrophages.There may were correlations with tviA,vex E and Nod2,Galectin8. |
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