Mechanism Of Salmonella Typhi Type ? Secretion System Affecting NF-?B Signaling Pathway In Macrophages

ObjectiveThe NF-?B signaling pathway is a vital pathway that enables cells to produce inflammatory effects.When Salmonella enterica serovar Typhi infects macrophages,it is able to activate the NF-?B signaling pathway through substances such as lipopolysaccharide and flagellin,produce inflammatory factors,and induce various inflammatory responses.Type VI secretion system is an extremely important secretion system,which can make a big difference in the intracellular survival and pathogenesis of bacteria by secreting effector proteins.Previous transcriptome from the laboratory indicated that Salmonella enterica serovar Typhi infection of macrophages was able to elicit m RNA level changes in genes involved in the NF-?B signaling pathway.In this study,we will verify it on the previous transcriptome,and further explore the relationship and mechanism between Salmonella enterica serovar Typhi type VI secretion system and NF-?B signaling pathway,which plays an important role in understanding the effect of Salmonella enterica serovar Typhi type VI secretion system on NF-?B signaling pathway and inflammation in Salmonella enterica serovar Typhi macrophages.Methods1.Analysis of Transcriptome from Macrophages infected by Salmonella enterica serovar Typhi(S.Typhi)The genes with statistical differences were selected by comparing 2 h,24 h transcription with0 h,and the functions of these genes were classified and enriched.2.Validation of results related to NF-?B signaling pathway by transcriptome analysisThe macrophage transcriptome results were verified by examining the expression levels of key genes and proteins of the NF-?B pathway in macrophages infected with S.Typhi for 2 h and24 h.(1)THP-1 cell culture and induction:Before the experiment,the cells were counted using a bovine abalone technical plate,the density of the cells reached 5×10~5 cells/m L after dilution,and macrophage infection experiments were performed after 48 h of induction using ethyl phorbol ester(PMA).(2)Cell infection test:wild strain of S.Typhi is used for cell infection test.Macrophage total RNA and total cellular protein were collected after 2 h and 24 h of infection using the time after 1h of gentamicin addition as the starting time point.3.Effect of S.Typhi on NF-?B Signaling Pathway in the Early Phase of Macrophage InfectionThe trend of NF-?B signaling pathway changes after different phases(including 2 h,4 h,6 h and short time 5 min,15 min,and 30 min)of S.Typhi infected macrophages was detected and analyzed.Infection assays were performed at an MOI of 20:1,and macrophage total RNA or total protein was extracted after culture for the corresponding time.For the short time 0-30 min test of macrophages infected with S.Typhi,there is no need to add gentamicin,and total RNA of macrophages or total protein of cells is directly extracted by adding bacterial infection until the corresponding time.4.Effect of Type VI Secretion System(T6SS)on NF-?B Signaling Pathway in MacrophagesTo detect and investigate the trend of NF-?B signaling pathway changes in macrophages infected with different strains of T6SS at different phases.The strains used in the experiment and the infection process were as follows:(1)?sciS:WT-p E,?sciS-p E as well as C-?sciS strains were used for macrophage infection experiments,and macrophage RNA or total cellular protein was extracted after 2 h,6 h,12 h and24 h of culture time.(2)High expression strains of sciK gene in T6SS:WT-p BAD/g III and WT-psciK strains were used for macrophage infection experiments,and macrophage RNA or total cellular protein was extracted after 2 h,6 h,12 h and 24 h of culture.5.T6SS affects the expression of downstream proinflammatory factors mediated by NF-?B signaling pathway(1)Changes in the expression of inflammatory factors such as TNF-?,IL-1?,and IL-6 after infection of macrophages with sciS-deficient strains of the T6SS and strains with high sciK gene expression were examined using q RT-PCR.(2)Using chromatin co-immunoprecipitation(Ch IP)-qPCR technology:Chromatin co-immunoprecipitation technology is an experimental approach to study protein interactions with DNA in vivo.This method was used to obtain the downstream target DNA of p65 binding after NF-?B complex(p65-p50)entry into the nucleus and NFKBIZ inhibitory protein binding in macrophages infected with sciS-deficient and sciK gene-highly expressing strains for 12 h and 24h,respectively,and then the expression of this inflammatory factor was quantitatively detected by qPCR technique.Results1.Transcriptome data analysisGenes related to the canonical pathway of the NF-?B signaling pathway,p50,p65,and genes such as p52 and rel B of the non-canonical pathway,were all up-regulated at 2 h or 24 h of macrophage infection,while transcript levels of negative regulators encoding the NF-?B signaling pathway such as Nfkbiz,Tnfaip3,Cyld,Tnip1,Tnip3,Traf1,and Zc3h12a were also increased at 2h or 24 h of infected.2.Validation of results related to NF-?B signaling pathway by transcriptome analysisQRT-PCR analysis of genes such as p50 and p65 in the canonical pathway as well as p52 and Relb in the non-canonical pathway and negative regulators encoding the NF-?B signaling pathway such as Nfkbiz,Tnfaip3,Cyld,Tnip1,Tnip3,Traf1 as well as Zc3h12a was generally consistent with the trend of transcriptome gene results.3.Effect of S.Typhi on NF-?B Signaling Pathway in the Early Phase of Macrophage Infection(1)0–30 min:The q RT-PCR results suggested that the m RNA levels of NF-?B-related genes were up-regulated in both S.Typhi infected macrophages at 30 min,such as the expression of Nfkbiz,Tnfaip3,Cyld,Tnip1,Tnip3,Traf1,and p50,which are negative regulators of the NF-?B signaling pathway,did not change significantly.(2)0-6 h:The m RNA level of p50 gene of canonical pathway of NF-?B signaling pathway was enhanced after 2 h–6 h infection,the change trend of non-canonical pathway was similar to that of canonical pathway inhibition,and the expression of NFKBIZ,an inhibitor protein of canonical pathway,was up-regulated at 2 h,4 h and 6 h after infection,and the non-specific inhibitors Tnfaip3,Cyld,Tnip1,Tnip3,and Traf1 were up-regulated after infection.4.Effect of T6SS on NF-?B signaling pathway in macrophages(1)?sciS:q RT-PCR results showed that p50 expression levels were decreased relative to WT when?sciS-infecting macrophages were cultured at 2 h in the early and 24 h in the late phase,with no significant difference at 6 h and 12 h.Western blot results showed that the phosphorylation level of p65 at 2 h in?sciS-infected macrophages was down-regulated compared with WT,the phosphorylation level of p65,a key protein of the canonical pathway,was significantly up-regulated compared with WT at 6 h,12 h and 24 h.The cleavage products p52 and Rel B of p100,a key protein of the non-canonical pathway,were up-regulated at 12 h and 24 h after infection.The m RNA levels of NFKBIZ,an inhibitor of the NF-?B signaling pathway,were down-regulated at 2 h,6 h,12 h and 24 h,and their protein expression levels were few vital difference,while the m RNA levels of TNIP1 and CYLD,inhibitors of the common pathway,were up-regulated at 12 h and 24 h.(2)High sciK expression:The q RT-PCR results indicated that p50,a key gene of the canonical pathway,was upregulated at both 2 h–24 h after infection of macrophages in sciK high expressing strains compared with WT.Westernblot results showed that the phosphorylation level of p65protein was significantly higher at 6 h and 24 h after infection,and its specific suppressor NFKBIZ m RNA expression level and protein expression level were also correspondingly up-regulated at 6h and 24 h.The major protein of the non-canonical pathway,p52,as well as was significantly up-regulated at 12 h and 24 h after infection,and Rel B protein expression levels were significantly up-regulated at 24 h after infection.Non-specific repressor CYLD m RNA expression levels were up-regulated at 2 h and 6 h after infection,and TNIP1 was up-regulated at both 2 h–24 h of infection.5.T6SS affects the expression of downstream proinflammatory factors mediated by NF-?B signaling pathway(1)q RT-PCR:Compared with WT-p BAD33,?sciS-p BAD33 significantly up-regulated the m RNA expression level of TNF-?at 6 h,but down-regulated at 12 h,up-regulated IL-1?at both the middle and late stages of infection,and up-regulated IL-6 at the late stage of infection in infected macrophages.The expression levels of IL-6 were up-regulated at 2 h–24 h after infection in both WT-p BAD/g III compared with WT-psciK.(2)The expression of inflammatory factors by Ch IP-qPCR:Binding of NF-?B to the TNF-?promoter was inhibited and binding to IL-1?and IL-6 was elevated after 6 h of infection in?sciS compared with WT.The sciK-high expressing strain was able to inhibit NF-?B binding to the TNF-?and IL-1?promoters and promote binding to the IL-6 promoter at 12 h after infection.Conclusions1.S.Typhi infection of macrophages is able to activate the NF-?B signaling pathway,but it can also initiate the expression of pathway-related inhibitors.2.The sciS gene of S.Typhi T6SS structural gene can stimulate the expression of canonical pathway and non-canonical pathway of this pathway in macrophages 2 hours after infection, but this pathway will be inhibited at 6 hours,12 hours and 24 hours after infection.3.Ch IP-qPCR results suggest that the sciS gene is able to act a specific part in impeding the expression of inflammatory factors induced by the canonical pathway,that is,the binding of NF-?B to the TNF-?promoter is limited and the binding to IL-1?and IL-6 is increased after 6 h of infection.4.sciK was able to promote the up-regulation of NF-?B signaling pathway,in which the level of canonical pathway was significantly higher at 6 h and 24 h after infection,and its specific inhibitor protein NFKBIZ expression level was also correspondingly up-regulated,which is likely to play an inhibitory role in NFKBIZ up-regulation after the activation of p65-p50 complex into the nucleus.5.Ch IP-qPCR results suggest that sciK is able to inhibit the binding of p65-p50 transcription factor to TNF-?and IL-1?promoters after NF-?B binding to NFKBIZ,while the binding to IL-6 promoter is up-regulated after 6 hours of infection.In addition,sciK was able to enhance the expression of non-canonical pathways at 12 h and 24 h after macrophages were infected.