The Influence Of Salmonella Virulence Genes SpvB On Macrophage Iron Homeostasis And Its Mechanisms

Objective:To investigate the mechanisms of Salmonella virulence genes spvB on bacterial pathogenic and the host immune,we firstly established Salmonella macrophages infection model to study the molecular mechanisms of spvB on macrophage iron homeostasis,and it will provide us new insight into the elucidating the spvB locus promotes intracellular multiplication of Salmonella,eventually leading to serious infection.Moreover,our research constructed human monocyte THP-1 cell lines with NLRP3 gene knocked out by the usage of CRISPR/Cas9 technique,which provide a tool for further investigate the cross-regulatory interactions between iron homeostasis and NLRP3 inducible immune effector pathways.Methods:One.The influence of Salmonella virulence genes spvB on macrophage iron homeostasis and its mechanisms1.The effect of Salmonella virulence gene spvB on bacterial growthWild-type S.typhimurium strain SR-11,spvB mutated strain SR-11-ΔspvB andΔspvB-complemented strain SR-11-c-ΔspvB were incubated at 37℃for 16 h in LB medium.The OD₆₀₀ of the cultures was monitored at interval of 16 h and the growth curves were measured.The dilutions of the cultures were spread on LB agar plates and incubated at 37°C before the CFU were counted.2.The influence of Salmonella virulence genes spvB on bacterial multiplication in macrophagesWild-type S.typhimurium strain SR-11,spvB mutated strain SR-11-ΔspvB andΔspvB-complemented strain SR-11-c-ΔspvB were used for infection.Macrophages were infected with strains which harvested as describled at a MOI of 20.Intracellular CFU analysis in macrophages with experimental strains for 4,8 or 12 h.In order to determine whether spvB promotes the intracellular multiplication of Salmonella is associated with intracellular iron content,bacterial burden（CFU）was determined 24 h post-infection following treatment with 100μM ferric ammonium citrate（FAC）or defriprone（DFP）.3.The effect of Salmonella virulence gene spvB on iron content in macrophagesPreparation of total RNA and cDNA was generated according the manufacturer’s protocol.Iron regulation related genes Fpn,TfR1 and DMT1 mRNA were measured by qPCR.Protein extracts were prepared with cytoplasmic lysis buffer.Ferroportin was determined by Western blotting.Intracellular iron levels were determined by atomic absorption spectrometry.4.The influence of Salmonella virulence genes spvB on NLRP3 in macrophagesNLRP3,IL-1βand IL-18 mRNA were measured by qPCR.NLR family pyrin domain containing 3（NLRP3）was determined by Western blotting.5.Iron related inflammatory factors were determined by qPCRThe cytokines genes Lcn2,TNF-α,IL-6 and IL-10 mRNA were measured by qPCR.Two.Establish NLRP3 knock out THP-1 cell strainsFour groups of sgRNA were designed to target the different exons of NLRP3 and cloning into pGL3 vector.Four recombinant plasmids were confirmed correct by Xho I or sequencing.Co-transfection the constructed recombinant plasmid vector and Cas9vector into human monocyte THP-1 cell lines.Screening the monoclonal cell strains with puromycin resistance via infiniting dilution procedure.Finally,identification the NLRP3 knock out cell strains by Western blotting.Results:One.The influence of Salmonella virulence genes spvB on macrophage iron homeostasis and its mechanisms1.The effect of Salmonella virulence gene spvB on bacterial growthThere were no significant difference in the growth of SR-11,SR-11-ΔspvB and SR-11-c-ΔspvB.2.The influence of Salmonella virulence genes spvB on bacterial multiplication in macrophagesPlate counting showed that SR-11 and SR-11-c-ΔspvB multiplied faster in infected macrophages than SR-11-ΔspvB infected group with the extension of infection time.These results indicated that spvB could promote intracellular multiplication of Salmonella.The number of Salmonella colony forming units（CFUs）recovered from macrophages after 24 h of infection was no significantly difference following pre-treatment with the ferric ammonium citrate or defriprone as compared to the solvent-treated control cells.It is suggested that the effect of spvB on bacterial multiplication in macrophages was related to the iron content.3.The effect of Salmonella virulence gene spvB on iron content in macrophagesAt all time points,we observed the ferroportin mRNA levels of SR-11-ΔspvB infection group was significantly higher than that of SR-11 and SR-11-c-ΔspvB infection groups,although cytoplasmic FPN m RNA levels were reduced at this time.However,The TfR1 and DMT1 expression following infection with S.Typhimurium that was no significantly difference in varies infection groups.To gain further insights into the dynamics of cellular iron trafficking,we determined intracellular iron concentrations via atomic absorption spectrometry.We observed that SR-11-ΔspvB infection group exhibited significantly lower iron contents following infection compared to other groups.4.The influence of Salmonella virulence genes spvB on NLRP3 in macrophagesNLRP3,IL-1βand IL-18 genes were significantly higher expressed in SR-11 and SR-11-c-ΔspvB infection groups than in SR-11-ΔspvB infection group.These results were paralleled by a significant increase which were determined by Western blotting.5.Iron related inflammatory factors were determined by qPCRThe mRNA levels of Lcn2,TNF-αand IL-6 in SR-11 and SR-11-c-Δspv B infected groups were significantly higher than those in SR-11-ΔspvB infection group and IL-10transcription level was lower than SR-11-ΔspvB infection group.Two.Establish NLRP3 knock out THP-1 cell strainsEstablish NLRP3 knock out THP-1 cell strains through CRISPR/Cas9 system.Western blotting identified two monoclonal cell strains with significantly declined NLRP3 protein expression level.It was showed that THP1-defNLRP3 cell strains was successfully constructed.Conclusions:1.The effect of spvB on bacterial multiplication in macrophages was related to the iron content.2.SpvB can inhibit the expression of FPN in macrophages and limit the efflux of intracellular iron,eventually leading to an increase in intracellular iron content.3.SpvB activates the NLRP3 inflammasome in macrophages,which is positively correlated with an increase in intracellular iron content.4.We constructed human monocyte THP-1 cell lines with NLRP3 gene knocked out by the usage of CRISPR/Cas9 technique,which provide a tool for further investigate the cross-regulatory interactions between iron homeostasis and NLRP3inducible immune effector pathways.