Regulation Of CpxR On Negative Regulatory Protein MgrB Of Salmonella Typhimurium

Salmonella typhimurium is an important zoonotic pathogen.With the widespread use of antibacterial drμgs in the prevention and treatment of Salmonella,Salmonella typhimurium has high resistance and multi-drμg resistance to clinically used drμgs(MDR).There are many kinds of two-component signal transduction systems(TCSs)in bacteria,which are closely related to the formation of bacterial drμg resistance and multiple drμg resistance(MDR).CpxAR system is one of the common TCS of negative bacteria,which plays an important role in regulating drμg resistance and MDR of Salmonella.It has been found that the pathways related to myxin resistance in Salmonella typhimurium are mediated by TCSs PhoPQ and PmrAB,and MgrB is a negative regulatory protein of PhoPQ.It was found that the insertion of IS5-like,IS1-like and ISKpn14 in Klebsiella pneumoniae resulted in the truncation or inactivation of mgrB and played an important role in myxin resistance.At present,no studies have found that cpxR regulates Salmonella mgrB in the absence of acrB.In this study,we investigated the regulatory effect of CpxR,a response regulator of Salmonella typhimurium TCS CpxAR,on the negative regulatory protein gene mgrB in the myxomycin resistance pathway,the regulation pathway of CpxR was analyzed to provide a new idea and experimental basis for further study of the interaction between different TCSs.Construction of Salmonella typhimurium mgrB single gene deletion strain(JSmgrB::kan abbreviated as JSA3::kan)by Red homologous recombination technique,the complete open reading frame of mgrB was amplified by PCR and cloned into prokaryotic expression vector pBAD/HisA,electrotransformation into JSAacrBAcpxR(abbreviated as JSΔ1Δ2)constructed in our laboratory,recombinant plasmid overexpression strain(JSΔ1Δ2/pmgrB);Construction of co-expression strains of cpxR and mgrB by Over-lapping PCR(JSΔ1Δ2/pcpxR-mgrB);using the technology of bacteriophage transduction,the previously constructed JSΔ1Δ2 was the recipient bacterium,and JSΔ3::kan was the donor bacterium,under the action of phage P22,acrB,cpxR and mgrB gene deletion strains and corresponding gene replenishment strains were constructed.Minimum inhibitory concentration(MICs)of myxomycin of each strain was determined by broth dilution method.Minimum inhibitory concentration(MICs)of myxomycin was determined by microbroth dilution method,and the concentration of myxomycin in LB broth and M9-0.2%glucose was also determined.The results showed that Salmonella typhimurium mgrB single gene deletion strain(JSΔ3::kan),mgrB overexpression strain(JSΔ1Δ2/pmgrB)and cpxR-mgrB co-expression strain(JSΔ1Δ2/pcpxR-mgrB),three gene deletion strain and corresponding gene replenishment strain were successfully constructed(JsΔ1Δ2A3::kan、JSΔ1Δ2Δ3::kan/pmgrB、JSΔ1Δ2Δ3::kan/pcpxR-mgrB、JSΔ1Δ2Δ3::kan/pcpxR).MIC results showed that of Colistin by JSΔ1Δ2/pccpxR-mgrB was 16,8,8 and 4 folds lower than that of JSΔ3::kan、JSΔ1Δ2Δ3::kan/pcpxR、JSΔ1Δ2Δ3::kan/pmgrB、JSΔ1Δ2Δ3::kan/pcpxR-mgrB,respectively;The MIC value of JS Delta 3::Kan to Colistin increased by 2 times than that of JS;the MIC value of JSΔ1Δ2Δ3::kan/pmgrB、JSΔ1Δ2Δ3::kan/pcpxR、JSΔ1Δ2Δ3::kan to Colistin did not change.The bactericidal curve test further proved that the bacterial activity of JSΔ3::kan、JSΔ1Δ2/pmgrB、JSΔ1Δ2Δ3::kan、JSΔ1Δ2Δ3::kanlpmgrB、JSΔ1Δ2Δ3::kan/pcpxR-mgrB、JSΔ1Δ2Δ3::kan/pcpxR、JSΔ1Δ2/pcpxR-mgrB strain was inhibited to varying degrees after incubation with different concentrations of Colistin in 30 min-12 h,after 8 h incubation with 12.8 mg/L colistin,the survival rate of these strains was less than 10%.Further analysis of the growth of each strain showed that JS、JSΔ1Δ2::kan、JSΔ3::kan、JSΔ1Δ2、JSΔ1Δ2/pmgrB、JSΔ1Δ2::kan/pcpxR、JSΔ1Δ2Δ3::kan、JSΔ1Δ2Δ3::kan/pmgrB、JSΔ1Δ2Δ3::kan/pcpxR-mgrB、J SΔ1Δ2Δ3::kan/pcpxR、JSΔ1Δ2/pcpxR-mgrB strain produced similar growth curve at 0-8 h in LB broth medium,but showed different endpoints at 14 h,JSΔ1Δ2/pcpxR-mgrB has the highest growth activity in LB broth(Compared with JS,P<0.003),JSΔ1Δ2Δ3::kan/pcpxR-mgrB has the minimum growth activity in LB broth(Compared with JSΔ1Δ2/pcpxR-mgrB,P<0.0001);Different phenomena were observed at 24 hours in M9-0.2%glucose medium,JSΔ1Δ2/pcpxR-mgrB has the Lower growth activity(Compared with JSΔ1Δ2Δ3::kan,p<0.00006),JSΔ1Δ2Δ3::kan has the highest growth activity(Compared with JSΔ1Δ2::kan p<0.01).These results sμggest that cpxR regulates the sensitivity of Salmonella typhimurium to colistin by regulating the expression of mgrB and downstream target genes in the colistin resistance pathway.