Regulation Of CpxR On Negative Regulatory Protein MgrB Of Salmonella Typhimurium

Salmonella typhimurium is an important zoonotic pathogen.With the widespread use of antibacterial dr?gs in the prevention and treatment of Salmonella,Salmonella typhimurium has high resistance and multi-dr?g resistance to clinically used dr?gs(MDR).There are many kinds of two-component signal transduction systems(TCSs)in bacteria,which are closely related to the formation of bacterial dr?g resistance and multiple dr?g resistance(MDR).CpxAR system is one of the common TCS of negative bacteria,which plays an important role in regulating dr?g 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 JS?1?2)constructed in our laboratory,recombinant plasmid overexpression strain(JS?1?2/pmgrB);Construction of co-expression strains of cpxR and mgrB by Over-lapping PCR(JS?1?2/pcpxR-mgrB);using the technology of bacteriophage transduction,the previously constructed JS?1?2 was the recipient bacterium,and JS?3::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(JS?3::kan),mgrB overexpression strain(JS?1?2/pmgrB)and cpxR-mgrB co-expression strain(JS?1?2/pcpxR-mgrB),three gene deletion strain and corresponding gene replenishment strain were successfully constructed(Js?1?2A3::kan?JS?1?2?3::kan/pmgrB?JS?1?2?3::kan/pcpxR-mgrB?JS?1?2?3::kan/pcpxR).MIC results showed that of Colistin by JS?1?2/pccpxR-mgrB was 16,8,8 and 4 folds lower than that of JS?3::kan?JS?1?2?3::kan/pcpxR?JS?1?2?3::kan/pmgrB?JS?1?2?3::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 JS?1?2?3::kan/pmgrB?JS?1?2?3::kan/pcpxR?JS?1?2?3::kan to Colistin did not change.The bactericidal curve test further proved that the bacterial activity of JS?3::kan?JS?1?2/pmgrB?JS?1?2?3::kan?JS?1?2?3::kanlpmgrB?JS?1?2?3::kan/pcpxR-mgrB?JS?1?2?3::kan/pcpxR?JS?1?2/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?JS?1?2::kan?JS?3::kan?JS?1?2?JS?1?2/pmgrB?JS?1?2::kan/pcpxR?JS?1?2?3::kan?JS?1?2?3::kan/pmgrB?JS?1?2?3::kan/pcpxR-mgrB?J S?1?2?3::kan/pcpxR?JS?1?2/pcpxR-mgrB strain produced similar growth curve at 0-8 h in LB broth medium,but showed different endpoints at 14 h,JS?1?2/pcpxR-mgrB has the highest growth activity in LB broth(Compared with JS,P<0.003),JS?1?2?3::kan/pcpxR-mgrB has the minimum growth activity in LB broth(Compared with JS?1?2/pcpxR-mgrB,P<0.0001);Different phenomena were observed at 24 hours in M9-0.2%glucose medium,JS?1?2/pcpxR-mgrB has the Lower growth activity(Compared with JS?1?2?3::kan,p<0.00006),JS?1?2?3::kan has the highest growth activity(Compared with JS?1?2::kan p<0.01).These results s?ggest 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.