| Objective:Iron is the necessary nutrient for the growth and metabolism of bacteria,and the increase of intracellular iron content is beneficial to the bacteria growth.Based on the discovery that Salmonella plasmid virulence gene spvB is closely related to cellular iron metabolism,this study will further explore the underlying molecular mechanism.Firstly,the infection model of RAW264.7 macrophage and MEF established to investigate the molecular mechanism of the effects of spvB on celluar iron metabolism,which provided us new insights for spvB in promoting the intracullular growth of host bateria.Secondly,the RAW264.7 macrophage that knockouted the nuclear transcription factor Nrf2 was contructed via CRISPR/Cas9 gene editing technique,which provided a tool for further investigate the effects of spvB on celluar iron metabolism.The infection models of RAW264.7 macrophage and Nrf2-/-RAW264.7 macrophage established to further explore the function of NRF2/FPN pathway in the effect of spvB on cellullar iron metabolism,which provided theoretical and experimental basis for the in-depth study of the pathogenesis of spvB,also for the development of new strategies for preventing and controlling Salmonella infection.Methods:One.Mechanism research on the effects of Salmonella plasmid virulence gene spvB on cellular iron metabolism which promoting the intracullular growth of host bateriaial1.The function of cellullar iron metabolism in Salmonella plasmid virulence gene spvB promoting the intracullular growth of host bateriaialCo-culture Wild-type S.typhimurium strain SL1344,spvB mutated strain SL1344-?spvB and ?spvB-complemented strain SL1344-c-spvB with the RAW264.7 murine macrophage at a MOI of 10 to establish infection model.At 2 h,4 h and 8 h post co-culture,t infected cells were lysed and enumerated colony-forming units to enumerate colony-forming units to analyze the effects of spvB on bateriaial growth.At the indicated time points,culture supernatants collected for the determination of LDH.Plates were analyze by measuring the absorbance at 490 nm to analyze the effects of spvB on cell injury.RAW264.7 murine macrophage supplemented with FAC,ferrous and DFP for 30 min and subsequently infected with S.Typhimurium at a MOI of 10.At 4 h post co-culture,infected cells were lysed and enumerated colony-forming units to confirm the effects of cellullar iron metabolism on spvB promoting bateriaial growth.2.Mechanism research on the effects of Salmonella plasmid virulence gene spvB on cellular iron metabolismCo-culture Wild-type S.typhimurium strain SL1344,spvB mutated strain SL1344-?spvB and ?spvB-complemented strain SL1344-c-spvB with the RAW264.7 murine macrophage at a MOI of 10 and with MEF at a MOI of 5 to establish infection model.At 2 h,4 h and 8 h post co-culture,the expression levels of TFRC,DMT1 and FPN were evaluated by qPCR and Western Blot to analyze the effects of spvB on iron metabolism related proteins.3.Mechanism research on the effects of Salmonella plasmid virulence gene spvB on FPNThe 5 ?M transcriptional inhibitor actinomycin D was added to the above cell infection model and the expression levels of Fpn were evaluated by qPCR at 2 h post co-culture to confirm wether spvB regulated FPN expression at transcriptional level.At 2 h,4 h and 8 h post co-culture,the expression levels of NRF2,HIF-1? and MTF1 were evaluated by qPCR and Western Blot to analyze the effects of spvB on FPN.Two.Mechanism research on the effects of Salmonella plasmid virulence gene spvB on cellullar iron metabolism via NRF2/FPN pathway1.Construct the Nrf2 knockout RAW264.7 murine macrophageThe Nrf2 knockout RAW264.7 murine macrophage was constructed through CRISPR/Cas9 system.One group of sg RNA were designed to target one of the exons of Nrf2 and cloning into p GL3 vector.Then the recombinant plasmid was confirmed by Xho I or sequencing.Co-transfection the p GL3-sg RNA recombinant plasmid and the Cas9 plasmid into h RAW264.7 murine macrophage.Screening the monoclonal cell strains with puromycin resistance via infiniting dilution procedure.Finally,NRF2 levels of monoclonal cell strains were evaluated by Western Blotting to confirm whether the Nrf2 knockout RAW264.7 was successfully constructed.2.The function of NRF2/FPN pathway in the effect of Salmonella plasmid virulence gene spvB on cellullar iron metabolismCo-culture the S.typhimurium strains mentioned above were with the RAW264.7 murine macrophage and Nrf2-/-RAW264.7 murine macrophage at a MOI of 10 to establish infection model.At 2 h post co-culture,the expression levels of Fpn were evaluated by qPCR to analyze the function of NRF2 in the effects of spvB on the transcription of FPN.At 4 h post co-culture,subcellular distribution of NRF2 and Hoechst were evaluated by immunofluorescence.At 4 h post co-culture,cytoplasmic protein and nucleoprotein were extracted and the expression levels of NRF2 were evaluated by Western Blot to analyze the mechanism of spvB inhibited the transcription of FPN.3.Effects of different domains of Salmonella plasmid virulence gene spvB on NRF2To further explore the effects of different domains of spvB on NRF2,co-culture the S.typhimurium strains mentioned above,spvB C-terminal mutated strain SL1344-spvB ?C-terminal and spvB N-terminal mutated strain SL1344-spvB ?N-terminal were with the RAW264.7 murine macrophage at a MOI of 10 to establish infection model.Western Blot evaluated the expression levels of NRF2 at 2 h post co-culture.Results:One.Mechanism research on the effects of Salmonella plasmid virulence gene spvB on cellular iron metabolism which promoting the intracullular growth of host bateriaial1.The function of cellullar iron metabolism in Salmonella plasmid virulence gene spvB promoting the intracullular growth of host bateriaialThe intracellular colony-forming units of SL1344 group and SL1344-c-spvB group were higher than that of the SL1344-?spvB group,which indicated that spvB promote the intracullular growth of host bateriaial.The content of LDH of SL1344 group and SL1344-c-spvB group were higher than that of the SL1344-?spvB group,which indicated that spvB promote cell injury.After supplemented with FAC,ferrous and DFP,the intracellular colony-forming units was no significantly difference between different groups,which indicated that the effect of spvB on bacteria growth was related to the cellullar iron metabolism.2.Mechanism research on the effects of Salmonella plasmid virulence gene spvB on cellular iron metabolismqPCR results show that the expression levels of Tfrc and Dmt1 has no significantly difference between different groups in the early stage of infection,which indicated that the effect of spvB on cellullar iron metabolism may not be related to the biological process of cellullar iron uptake.But qPCR and Western Blot results show that the expression levels of FPN in SL1344 group and SL1344-c-spvB group were lower than that of the SL1344-?spvB group.The results mentioned above indicated that spvB might inhibit the expression of FPN and limit the efflux of intracellular iron,which eventually leading to an increase of intracellular iron content.3.Effects of different domains of Salmonella plasmid virulence gene spvB on NRF2qPCR results show that the levles of Fpn has no significantly difference between different groups after treatment with actinomycin D at 2 h post co-culture,which indicated that spvB regulated FPN expression at transcriptional level.qPCR results show that undiscovering the expression level of Hif-1? and Mtf1 in the SL1344 group and SL1344-c-spvB group were lower than that of the SL1344-?spvB group,which indicated that the effect of spvB on cellullar iron metabolism may not be related to HIF-1? and MTF1.But qPCR and Western Blot results show that the expression levels of NRF2 in SL1344 group and SL1344-c-spvB group were lower than that of the SL1344-?spvB group.The results mentioned above indicated that spvB down regulate the transcription level of FPN by inhibiting the expression of NRF2.Two.Mechanism research on the effects of Salmonella plasmid virulence gene spvB on cellullar iron metabolism via NRF2/FPN pathway1.Construct the Nrf2 knockout RAW264.7 murine macrophageColony PCR identification results show the recombinant vector of sg RNA was successfully constructed.The results of enzyme digestion and DNA sequencing show that the recombinant plasmid of p GL3-sg RNA was successfully constructed.Western blot results confirm that the Nrf2 knockout RAW264.7 murine macrophage was successfully constructed.2.The function of NRF2/FPN pathway in the effect of Salmonella plasmid virulence gene spvB on cellullar iron metabolismqPCR results show that the expression levels of Fpn has no significantly difference between different groups in the infection model of Nrf2-/-RAW264.7,which indicated that spvB might affect cellullar iron metabolism through NRF2/FPN pathway.Immunofluorescence results show that the subcellular distribution of NRF2 in the nucleus of the SL1344 group and the SL1344-c-spvB group were lower than that of the SL1344-?spvB group.Western blot results show that the expression levels of Fpn in the Cytoplasm and nucleus of the SL1344 group and the SL1344-c-spvB group were lower than that of the SL1344-?spvB group.The results mentioned above indicated that spvB inhibit the transcription of FPN by blocking the entry of NRF2 into the nucleus.3.Effects of different domains of Salmonella plasmid virulence gene spvB on NRF2Western blot results show that the expression levels of NRF2 in SL1344-spvB ?N-terminal group was lower than that of the SL1344-?spvB group and had no significantly difference between that of the SL1344 group and SL1344-c-spvB group.However,the expression levels of NRF2 in SL1344-spvB ?C-terminal group had no significantly difference between that of the SL1344-?spvB group,but it was higher than that of the SL1344 group and the SL1344-c-spvB group.This result indicated that the function of spvB to inhibit NRF2 activation might mainly mediated by the carboxyl terminal domain of spvB.Conclusions:1.Salmonella plasmid virulence gene spvB plays a significant role in promoting intracellular growth of host bacteria by affecting the cellullar iron metabolism.2.Salmonella plasmid virulence gene spvB inhibits the expression of FPN by blocking the entry of NRF2 into the nucleus,which deregulates intracellular iron efflux and eventually leads to an increase of intracellular iron content.3.The function of Salmonella plasmid virulence gene spvB to inhibit NRF2 activation might mainly mediated by the carboxyl terminal domain of spvB. |
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