| Objective:As the intestinal pathogenic bacteria,Salmonella can infect humans and animals in fecal-oral route,causing Salmonellosis even death.Most of pathogenic Salmonella contain Salmonella plasmid virulence(spv)and spvB locus encoded protein plays an important role in Salmonella infection.As the most abundant cells in peripheral leukocytes,neutrophils arrive to the infected sites in first time and work in clearing bacteria by phagocytosis,the release of cytokines and the formation of neutrophil extracellular trap(NET).NET is a network structure based on DNA which covered with protease.To explore the mutual interaction of anti-Salmonella infection immunity between spvB operon and NETs,the first part of this research observed the influence of spvB on extracellular trap(ET)and NETs in vivo,using Salmonella enterica serovar Typhimurium(STM)wild type strain SL1344 and spvB locus delete mutation SL1344-?spvB to infect zebrafish larvae with only the innate immunity.In the second part,by co-culturing SL1344,SL1344-?spvB and spvB locus complementary strain SL1344-c-spvB with neutrophils,infection cell models were established to investigate the effect of spvB operon on NETs formation and its molecular mechanism.Study results would provide evidences in theory and experiment to illuminate the pathogenic mechanisms of spvB locus,as well as the functions of neutrophils in anti-infection immunityMethods:One.Observing the impact of spvB locus on ETs and NETs formation in zebrafish larvae1.The effect of spvB operon on ETs formation in zebrafish larvaeZebrafish larvae in 5 days post fertilization(dpf)were infected with 109 CFU/ml SL1344 and SL1344-?spvB by immersion,respectively.The larvae were collected at 6,8 and 10 hours post infection(hpi),the expression of citrullinated histone 3(CitH3)and peptidyl arginine deiminase-4(PAD4)were detected by Western Blot2.The establish of bacteria carrying red fluorescent protein(RFP)markersThe plasmid which expressed RFP was electroporated into SL1344 and SL1344-?spvB.After screening of Ampicillin(Amp)plates,the growing strains were observed by fluorescent microscope and verified by ploymerase chain reaction(PCR)amplifications.3.The effect of spvB operon on NETs formation in zebrafish larvaeTransgenic zebrafish larvae Tg(lyz:GFP)with green fluorescent protein(GFP)marked neutrophils at 5 dpf were infected with 109 CFU/ml RFP-SL1344 and RFP-SL1344-?spvB by immersion,then zebrafish larvae were inmersed in the water with SYTOX Blue for 30 min before collected.The larvae were collected at 8 hpi and the formation of NETs in vivo were observed by cell imager.Two.The influence of spvB locus on NETs formation and its molecular mechanism1.The establishment of neutrophil infection modelThe steadily growing human promyelocyte HL-60 cells were induced with 1.25%dimethyl sulfoxide(DMSO)and were collected at 6,7 and 8 days post induction(dpi).The expression of CD11b in differentiated HL-60(dHL-60)cells was detected by flow cytometry(FCM),and the cellular morphology after Wright Giemsa staining was observed under optical microscope.SL1344 was co-cultured with dHL-60 at multiplicity of infection(MOI)of 5:1,10:1 and 20:1.Samples were collected at 2 hpi and observed by optical microscope after fixed with methanol and stained by Wright Giemsa.SL1344 was co-cultured with dHL-60 at MOI of 10:1 and cells were collected at 1 hpi,2 hpi,4 hpi,6 hpi and 8 hpi.The expression of CitH3 in collected cells was detected by Western Blot.2.The effect of spvB operon on NETs formationSL1344,SL1344-?spvB and SL1344-c-spvB were co-cultured with dHL-60 at MOI of 10:1.At 2 hpi,extracellular DNA structures were observed under fluorescent microscope after treated with 70 U/ml deoxyribonuclease(DNase)for 15 min and stained with SYTOX Green.Three strains were co-cultured with dHL-60 at MOI of 10:1 and samples were collected in 1 hpi,2 hpi and 4 hpi.The expression of myeloperoxidase(MPO),CitH3 and PAD4 were detected by Western Blot.The concentration of double-strand DNA(dsDNA)in supernatant was detected by Picogreen assay.3.The molecular mechanism of NETs formation affected by spvB locusSL1344,SL1344-?spvB and SL1344-c-spvB were co-cultured with dHL-60 at MOI of 10:1.Collected at 1 hpi,2 hpi and 4 hpi,the expression of phosphorylation protein kinase C(p-PKC)was determined by Western Blot.With or without the pretreatment of protein kinase C(PKC)inhibitor,chelerythrine(CI),at the concentration of 1 ?M,2 ?M,5 ?M,10 ?M and 20 ?M for 30 min,dHL-60 cells were infected with SL1344 at MOI of 10:1.At 2 hpi,the expression of p-PKC and PAD4 in collected cells were determined by Western Blot.With or without pretreatment of CI at the concentration of 20 ?M for 30 min,dHL-60 cells were infected with three strains mentioned above at MOI of 10:1,and collected at 2 hpi.NETs formation was observed under fluorescent microscope after stained with SYTOX Blue.The expression of p-PKC,MPO,CitH3 and PAD4 were determined by Western Blot.The concentration of dsDNA in supernatant was detected by Picogreen assayResults:One.Observing the impact of spvB locus on ETs and NETs formation in zebrafish larvae1.The effect of spvB operon on ETs formation in zebrafish larvaeZebrafish larvae at 5 dpf were infected SL1344 and SL1344-dspvB by immersion,the expression of CitH3 and PAD4 were detected by Western Blot.The CitH3 levels of SL1344-?spvB infection group were higher than that in SL1344 infection group at 6 hpi,8 hpi and 10 hpi(P<0.05).The PAD4 levels of SL1344-?spvS infection group were higher than SL1344 infection group at 6 hpi and 8 hpi(P<0.05),while the PAD4 levels of the two infection groups had no significant difference at 10 hpi.These results suggested that spvB locus can inhibit ETs formation in zebrafish larvae.2.The establish of bacteria carrying RFP markersSL1344 and SL1344-?spvB were electroporated with plasmid expressed RFP,and both of RFP-SL1344 and RFP-SL1344-?spvB were screened by Amp plates.The gained colonies present red fluorescence under fluorescent microscope.According to the results of PCR,RFP-SL1344 contains spvB,spvC and rfp genes,RFP-SL1344-?spvB contains spvC and rfp genes.3.The effect of spvB operon on NETs formation in zebrafish larvaeTransgenic zebrafish larvae Tg(lyz:GFP)with GFP marked neutrophils at 5 dpf were infected with RFP-SL1344 and RFP-SL1344-?spvB by immersion and stained with SYTOX Blue by immersion,the NETs formation was observed by cell imager.Results showed that NETs structure,which means neutrophils marked by GFP were merged with extracellular DNA structure stained by SYTOX Blue and bacteria marked by RFP,emerged in both of RFP-SL1344 and RFP-SL 1344-?spvB infection group.And RFP-SL1344-?spvB infection group showed more NETs structures than RFP-SL1344 infection group.Two.The influence of spvB locus on NETs formation and its molecular mechanism1.The establishment of neutrophil infection modelHL-60 cells were induced by 1.25%DMSO for 6,7 and 8 d and the expression of CD11b was determined by FCM by then.Results showed that the CD11b levels were highest among three days which surpassing 80%at 7 dpi,and cells present classical neutrophil morphology after Wright Giemsa staining.Above all,using dHL-60 at 7 dpi for post experiments.SL1344 were co-cultured with dHL-60 at an MOI of 5:1,10:1 and 20:1 for 4 h.Wright Giemsa staining showed that Salmonella entered into all cells in every group.The cellular structure is intact at an MOI of 5:1 which is similar with the uninfected group.Only a few cells cytoplasm becomed looser at an MOI of 10:1.At an MOI of 20:1,the structure of partial cells were obviously destroyed.Above all,using an MOI of 10:1 for post experiments.SL1344 were co-cultured with dHL-60 at the MOI of 10:1,and cells were collected at 1 hpi,2 hpi,4 hpi,6 hpi and 8 hpi.The CitH3 levels of SL1344 infection group determined by Western Blot were higher than uninfected group at all detected points(P<0.05).The CitH3 levels of SL1344 infection group show no significant difference among 4 hpi,6 hpi and 8 hpi.Above all,using 1 hpi,2 hpi and 4 hpi as detected points for post experiments.2.The effect of spvB operon on NETs formationSL1344,SL1344-?spvB and SL1344-c-spvB were co-cultured with dHL-60 at the MOI of 10:1 for 2 h following with or without the treatment of DNase.Without the treatment of DNase,the extracellular DNA network structures of SL1344-?spvB infection group were more than SL1344 infection group and SL1344-c-spvB infection group.After the treatment of DNase,the extracellular DNA network structures disappeared in all of three infection groups.SL1344,SL1344-?spvB and SL1344-c-spvB were co-cultured with dHL-60 at the MOI of 10:1 and cells were collected at 1 hpi,2 hpi and 4 hpi.At 1 hpi,2 hpi and 4 hpi,the MPO,CitH3 and PAD4 levels of SL1344-AspvB infection group detected by Western Blot were higher than SL1344 infection group and SL 1344-c-spvB infection group(P<0.05).The dsDNA concentrations in supernatant of SL1344 infection group and SL1344-c-spvB infection group detected by Picogreen assay were lower than SL 1344-?spvB group(P<0.05)at 1 hpi and 2 hpi.While at 4 hpi,the dsDNA concentration of the three infection groups had no significant difference.These results suggested that spvB locus can inhibit NETs formation.3.The molecular mechanism of NETs formation affected by spvB locusSL1344,SL1344-?spvB and SL1344-c-spvB were co-cultured with dHL-60 at the MOI of 10:1,and cells were collected at 1 hpi,2 hpi and 4 hpi.The p-PKC levels of SL1344-AspvB infection group determined by Western Blot were higher than SL1344 group and SL1344-c-spvB group at 1 hpi and 2 hpi(P<0.05),while at 4 hpi,the p-PKC levels of the three infection groups had no significant difference.With the pretreatment of CI at the concentration of 1 ?M?2 ?M?5 ?M?10 ?M and 20 ?M for 30 min,dHL-60 were co-cultured with SL1344 for 2 h at the MOI of 10:1 and determined by Western Blot.The p-PKC levels of CI pretreated group at 20 ?M were significantly lower than CI non-pretreated group(P<0.001),and the PAD4 levels of CI pretreated groups at 5 ?M,10 ?M and 20 ?M were significantly lower than Cl non-pretreated group(P<0.001).Above all,using the concentration of CI at 20 ?M for post experiments.With the pretreatment of CI at 20 ?M for 30 min,dHL-60 were co-cultured with SL1344,SL1344-?spvB and SL1344-c-spvB at the MOI of 10:1 for 2 h.The results from fluorescent microscope showed that the extracellular DNA network structures disappeared in all of three infection groups of CI pretreated group.Western Blot showed that the p-PKC,MPO,CitH3 and PAD4 levels in CI pretreated group were significantly lower than CI non-pretreated group(P<0.001).The concentration of dsDNA in supernatant detected by Picogreen assay were significantly lower in CI pretreated group than CI non-pretreated group(P<0.01).These results showed that spvB locus can affect NETs formation by inhibiting the expression of p-PKC and PAD4.Conclusions:1.spvB locus can inhibit ETs and NETs formation in zebrafish larvae2.spvB locus can inhibit NETs formation in neutrophil3.spvB locus can affect NETs formation by inhibiting the expression of p-PKC and PAD4... |
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