Study The Underlying Mechanisms Of Macrophage Pyroptosis Suppressed By Salmonella Plasmid Virulence Gene SpvC

Objective:Salmonella plasmid virulence gene spvC,a phosphothreonine lyase,suppressed intestinal inflammation and increased bacterial dissemination through MAPK(mitogen-activated protein kinase)signaling pathway.Our previous study showed that effect was closely related to its inhibition of pyroptosis of macrophages.This study aims to investigate the underlying mechanisms of spvC suppression the pyroptosis of macrophages,which would provide new targets for controlling Salmonella infection and novel insights for the treatment of other corresponding diseases.We firstly co-cultured Salmonella typhimurium(S.typhimurium)with macrophages as infection models.In this part,we elucidated the relationship between spvC suppressing the pyroptosis of macrophages and MAPK signaling pathway,expression of NLRP3 and NLRC4 as well as oligomerization of apoptosis-associated speck-like protein containing a CARD(ASC).In the second part,CRISPR/Cas9 system was used to knock out the key protein gasdermin D(GSDMD)of pyroptosis in RAW 264.7 cells.gsdmd-/-RAW 264.7 cell would be used as a tool to study the effects and mechanisms of innate immune response and antibacterial function in macrophages mediated by GSDMD.Methods:Part one:The correlation between suppression of macrophagy pyroptosis by Salmonella plasmid virulence gene spvC and MAPK signaling pathway as well as inflammasomesIn this part,three bacterial strains,S.typhimurium wild type strain SL1344(STM-WT)together with spvC locus deletion-mutant and complementary strains(STM-?spvC and STM-c-spvC)that contrusted by our group,were co-cultured with murine macrophage line J774A.1 and RAW 264.7 cells at the multiplicity of infection(MOI)of 10:1.The infected macrophages were collected at different time points for experiments.1 The effect of spvC on the ultrastructure of macrophagesIt was found that spvC could affect the morphology of Salmonella infected macrophages by Wright Giemsa staining and Live-cell imaging in our previous study.On this foundation,STM-WT,STM-?spvC and STM-c-spvC were co-cultured with J774A.1 cells for 16 h and 24 h,respectively.Ultrastructures of different infected groups were observed by transmission electron microscope(TEM)after preparing ultra-thin section.2 The effect of spvC on MAPK signaling pathway in macrophages and its relationship with NLRP3 and NLRC42.1 The effect of Salmonella plasmid virulence gene spvC on MAPK signaling pathwayIt was reported that purified SpvC inactivated extracellular regulated protein kinase(Erk)and c-Jun N-terminal kinase(JNK)in macrophages.To explore the effect of spvC on MAPK signaling pathway in Salmonella infected macrophages,STM-WT,STM-?spvC and STM-c-spvC were co-cultured with J774A.1 cells.Phosphorylation of the key proteins of MAPK signaling pathway named ERK1/2,JNK1/2 and p38 were analyzed by western blot after extracting total proteins.2.2 The effect of Salmonella plasmid virulence gene spvC on the activation of NLRP3 and NLRC4Our previous study found spvC could inhibit the expression of NLRP3 and NLRC4 of Salmonella infected macrophages in the late stage.To further explore the biological effects of spvC in the early stage of macrophage infection,J774A.1 cells were infected with Salmonella strains for 2 h,4 h and 8 h after cultured overnight to logarithmic phase.NLRP3 and NLRC4 were analyzed by western blot after extracting total proteins.2.3 The effect of Salmonella plasmid virulence gene spvC on MAPK signaling pathway and its relationship with NLRP3 and NLRC4To explore spvC suppressed NLRP3,NLRC4 and its relationship with MAPK signaling pathway,J774A.1 cells were co-cultured for 8 h with three bacterial strains after pre-treated with ERK inhibitors,JNK inhibitors and p38 inhibitors for 3 h.Macrophages untreated with inhibitors were served as controls.NLRP3 and NLRC4 were analyzed by western blot after extracting total proteins.3 Relationship between spvC suppressing pyroptosis of macrophages and ASC which associated with canonical inflammasome activation pathwayIt has been demonstrated that the construction of NLRP3 inflammasome was dependent on ASC and could mediate pyroptosis through both canonical and non-canonical inflammasome activation pathways,while the NLRC4 inflammasome could only mediate pyroptosis through canonical inflammasome activation pathway,and was partially ASC-dependent.This section was carried out in order to further clarify the role of NLRP3,NLRC4 and ASC in spvC suppressed pyroptosis.3.1 The effect of Salmonella plasmid virulence gene spvC on ASC3.1.1 Determination of transcription level of asc and ASC protein monomer by qPCR and western blotSTM-WT,STM-?spvC and STM-c-spvC were co-cultured with J774A.1 cells for 2 h,8 h,16 h and 24 h respectively.Transcription level of asc was analyzed by qPCR while ASC monomer was analyzed by western blot after extracting total proteins.3.1.2 Analysis of ASC foci imaging with fluorescence microscopeSTM-WT,STM-?spvC and STM-c-?spvC were co-cultured with J774A.1 cells.ASC foci were observed under fluorescence microscope after stained with FITC and Hochest.3.2 The effect of Salmonella plasmid virulence gene spvC on ASC-deficient macrophages3.2.1 ASC of RAW 264.7 and J774A.1 cells were analyzed by western blotASC monomer was analyzed by western blot after extracting total proteins from RAW 264.7 amd J774A.1.3.2.2 Determination of the expression of pyroptosis-related proteins GSDMD and GSDMD-NT by western blotSTM-WT,STM-?spvC and STM-c-spvC were co-cultured with RAW 264.7 cells for 2 h,4 h,8 h,12 h,16 h,20 h and 24 h,respectively.GSDMD and GSDMD-NT of supernatant and cell lysate from RAW 264.7 were analyzed by western blot after extracting total.Part two:Construction of gsdmdd-/-RAW 264.7 cells by CRISPR/Cas9 system1.Construction and identification of gsdmd-/-RAW 264.7 cells1.1 Construction of recombinant vector pGL3-sgRNAAccording to the principle of single guide RNA(sgRNA)of CRISPR/Cas9 system,four sgRNAs of the third exon of gsdmd were designed.The DNA fragment containing sgRNAs and U6 promoter from pUC57 was amplified by PCR.Both PCR products and vector pGL3 were digested by BsmSI and then ligated by T4 DNA ligase.Recombinant vector pGL3-sgRNA was transformed into DH5a,subsequently determined by PCR amplification and sequencing.1.2 Construction and identification of RAW 264.7-Cas9 cellsCas9 plasmid was transfected into RAW 264.7 cells by electroporation.The transcription level of cas9 was identified by qPCR after passed on three times.1.3 Construction and identification of gsdmd-/-RAW 264.7 cellsRecombinant vector pGL3-sgRNA was transfected into RAW-Cas9 cells by electroporation.Gsdmd gene were identified by PCR amplification and sequencing.The expression of GSDMD were analyzed by western blot.2 The effect of spvC on RAW 264.7 cell death mediated by GSDMDWild type and gsdmd-/-RAW 264.7 cells were co-cultured with STM-WT,STM-?spvC and STM-c-spvC for 24 h.To elucidate the relationship between macrophage death mediated by spvC and GSDMD,the release level of lactate dehydrogenase(LDH)was detected by colorimetry.Results:Part one:The correlation between suppression of macrophagy pyroptosis by Salmonella plasmid virulence gene spvC and MAPK signaling pathway as well as inflammasomes1 The effect of spvC on the ultrastructure of macrophagesImages of TEM showed that the proliferation of Salmonella in J774A.1 cells increased with time,and cell structure was destroied.At the late stage of 16 h and 24 h post infection,STM-AspvC infected macrophages were severely damaged that cytoplasm loosen,cell contents exflux and the pore-induced intracellular traps(PITs)with membrane-bound were observed.These results suggested spvC could affect the formation of PITs.2 The effect of spvC on MAPK signaling pathway in macrophages and its relationship with NLRP3 and NLRC42.1 The effect of Salmonella plasmid virulence gene spvC on MAPK signaling pathwayWestern blot results showed that the levels of phospho-ERK1/2,phospho-JNKl/2 and phospho-p38 were lower in J774A.1 cells infected with Salmonella carrying spvC than that infected with STM-?spvC at 16 h.These results demonstrated that Salmonella plasmid virulence gene spvC could inhibit MAPK signaling pathway.2.2 The effect of Salmonella plasmid virulence gene spvC on the activation of NLRP3 and NLRC4Western blot results showed that the levels of NLRP3 and NLRC4 were lower in J774A.1 cells infected with Salmonella carrying spvC than that infected with STM-?spvC at 8 h.These results indicated spvC could inhibit the activation of NLRP3 and NLRC4.2.3 The effect of Salmonella plasmid virulence gene spvC on MAPK signaling pathway and its relationship with NLRP3 and NLRC4Western blot results showed that the levels of NLRP3 and NLRC4 were lower in J774A.1 cells infected with Salmonella carrying spvC than that infected with STM-vspvC among groups untreated with MAPK inhibitors.The levels of NLRP3 and NLRC4 in J774A.1 cells decreased after pretreated with inhibitors.These results indicated that spvC inhibited the pyroptosis-related protein NLRP3 and NLRC4 through MAPK signaling pathway.3 Relationship between inhibition of spvC on pyroptosis and ASC which associated with canonical inflammasome activation pathway of macrophages3.1 The effect of Salmonella plasmid virulence gene spvC on ASC3.1.1 Determination of transcription level of asc and ASC protein monomer by qPCR and western blotResults showed high transcription level of asc at the early stage from 2 h to 8 h.Transcription level of asc was significantly higher in J774A.1 cells infected with Salmonella carrying spvC than that infected with STM-?spvC.However,there was no significant difference in the protein level of ASC monomer in each group.3.1.2 Analysis of ASC foci imaging with fluorescence microscopeResults showed that ASC foci were formed in macrophages infected with Salmonella.However the difference was not significant in the amount of ASC foci among Salmonella infected groups.These results indicated that ASC oligomerization in macrophages could be activated by Salmonella infection independent of spvC.3.2 The effect of Salmonella plasmid virulence gene spvC on ASC-deficient macrophages3.2.1 ASC of RAW 264.7 and J774A.1 cells were analyzed by western blotWestern blot results confirmed that ASC was not expressed in RAW 264.7 cells while it was normal in J774A.1 cells.3.2.2 Determination of the expression of pyroptosis-related proteins GSDMD and GSDMD-NT by western blotResults showed that the level of GSDMD-NT in RAW 264.7 cells increased with time in each infected group,which was lower both in cell lysate and supernatant in macrophages infected with Salmonella carrying spvC than that infected with STM-?spspvC.Difference was significant at the late stage of infection.In consideration of the absence of ASC expression in RAW 264.7 cells,results suggested that spvC suppressed pyroptosis of macrophage independent of ASC.It is known that NLRP3 mediated pyroptosis is relied on ASC.Therefore we speculated that the effect of spvC on pyroptosis of macrophages through canonical inflammasome activation pathway might involve in NLRC4.Part two:Construction of gsdmd-/-RAW 264.7 cells by CRISPR/Cas9 system1 Construction and identification of gsdmd-/-RAW 264.7 cells1.1 Construction of recombinant vector pGL3-sgRNARecombinant vector pGL3-sgRNA was constructed after determined by PCR and sequencing.1.2 Construction and identification of RAW 264.7-Cas9 cellsqPCR results showed that RAW 264.7 cells could express after electroporation.1.3 Construction and identification of gsdmd-/-RAW 264.7 cellsPCR amplification and sequencing results demonstrated that the third exon of gsdmd of RAW 264.7 cells was successfully knocked out.Western blot results confirmed GSDMD protein was not expressed in RAW 264.7 cells.These results showed gsdmd-/-RAW 264.7 cells were successfully constructed.2 The effect of spvC on RAW 264.7 cell death mediated by GSDMDThe results of colorimetry showed that the LDH release level was significantly higher in RAW 264.7 cells infected with Salmonella carrying spvC than that infected with STM-?spvC.However,LDH release level in each infected group was significantly decreased after gsdmd gene knocked out.These results indicated that spvC could inhibit macrophage death caused by Salmonella infection through GSDMD dependent pathway.Conclusion:1.Salmonella plasmid virulence gene spvC could affect PITs formation in macrophages.2.Salmonella plasmid virulence gene spvC inhibited the expression of NLRP3 and NLRC4 through MAPK signaling pathway.3.The effect of Salmonella plasmid virulence gene spvC on canonical pyroptosis pathway of macrophages might involve in the suppression of NLRC4 through ASC independent manner.4.gsdmd-/-RAW 264.7 cell was successfully constructed and would play a major role in further study of macrophage death and antibacterial function mediated by GSDMD.