Research On Regulatory Mechanism Of CpxA On AGAs And ?-lactams Resistance In Salmonella Enterica Serovar Typhimurium

Salmonella enterica serovar Typhimurium is an important pathogen which is involved in the public health.The strains of multidrug-resistant(MDR)salmonella typhimurium have been emerging and spreading due to the use of antibiotics.The bacteria have to evolve many sensory signaling systems to adapt the adverse environments,among them,the most typical example is bacterial two-component Systems(TCSs).The CpxRA two-component system is composed of the sense kinase CpxA and cytosolic response regulator CpxR.It is an important MDR regulation system in Salmonella enterica serovar Typhimurium.Deletion or overexpression of CpxR can cause changes in the resistance of Salmonella enterica serovar Typhimurium to aminoglycosides and ?lactam antibiotics.However,no systematic studies have been reported on the resistance regulatory mechanism of CpxA to Salmonella enterica serovar Typhimurium to date.The purpose of this study was to elucidate the mechanism of CpxA regulation of multidrug resistance to Salmonella enterica serovar Typhimurium.The cpxA deletion mutant strain JS?cpxA was constructed by the Red homologous recombination system and the MICs values of aminoglycosides(Amikacin,Netilmicin,Streptomycin and Kanamycin)and beta-lactam antibiotic antibiotics(Cefthiophene,cefuroxime,ceftriaxone sodium and cefotaxime)were determined to analyze its influence on drug resistance regulation.The cpxRA double mutant was then constructed to determine whether CpxA-induced drug resistance regulation was dependent on CpxR.The cpxA-acka-pta three-deletion mutant was constructed to analyze whether the drug resistance regulation caused by cpxA deletion depends on the Acka-Pta pathway.Compared with wild strains JS,the different antibiotic MICs value of JS?cpxA increase 2 to 8 times.Compared with JS ? cpxRA,the antibiotic MICs value of JS?cpxA increase 8 to 16 times.Compared with JS?cpxA?ackA-pta,the antibiotic MICs value of JS?cpxA increase 2 to 16 times.These results indicated that the absence of cpxA could lead to increased drug resistance in Salmonella enterica serovar Typhimurium,which depended on the activation of CpxR by the Acka-Pta pathway.To investigate the target genes involved in resistance regulation of cpxA deletion Salmonella enterica serovar Typhimurium to aminoglycosides and ?-lactam antibiotics.JS,JSJcpxA and JSJcpxR strains were used to transcriptome sequencing.The characteristics of differences expression genes,and GO and KEGG annotation and clustering analysis were performed.The transcriptome data of candidate target genes were verified by Q-PCR.The corresponding target gene deletion strains were constructed based on CpxA deletion strains,and the changes in MICs of Amikacin,Tobramycin,Cephalothin and Cefuroxime were measured.Based on the opposite drug resistance phenotype and the Q-PCR verification results,16 candidate genes cpxR include cpxP,degP,stm3030 et al.were selected.The MICs results of the candidate genes and cpxA double deletion strain showed that except the deletion of cpxR and cpxP,the positive and feedback genes of the CpxRA two-component system and other genes had no effect on drug resistance regulation of the CpxRA two-component system.This imply that the CpxRA two-component system regulates the resistance of emerica serovar Typhimurium as a composite effect.To study key sites of the CpxA and CpxR involved in the drug resistance regulation.The improved genetic modification strategy combined an intracellular recombination system(X Redmediated recombination),counterselection marker sacB,and seamless assembly in vitro was used to simultaneously construct the multiple variations at cpxA and cpxR.Only using the Red homologous recombination system,the method can easily and efficiently integrate different cpxA and cpxR gene modifications constructed on plasmids into the corresponding genome.The strains JScpxAL38F,JScpxA?92-104,JScpxAC-3×FLAG,JScpxRD51A,JScpxRM199A,JScpxRD51A/M199A,JScpxRSD and JScpxRN-3xFLAG were constructed using this method.Then the resistance regulation and the related mechanism of different cpxA modified strains(JS?cpxA,JSCpxAL38F,JSCpxA?92-104)to aminoglycosides(AGAs)and ?-lactam antibiotics was determined.The construction of JS?cpxARD51A and S?cpxARM199A were used to confirm whether the absence of cpxA regulates drug resistance depends on the key functional sites of CpxR.The results showed that both D51A and M199A mutations in CpxR could effectively inhibit the increase of drug-resistance caused by cpxA deletion.The JS?acrB,JS?cpxA?acrB,JScpxA38?acrB,JScpxA92-104?acrB,JS?tolC,JS?cpxA?tolC,JScpxA38?tolC and JScpx92-104?tolC were constructed to confirm effect of efflux pump AcrAB-TolC on the regulation of drug resistance of these CpxA modified strains.The results indicated that all three cpxA mutations upregulated the MICs value of the tested antibiotics independent of the gene acrB or tolC.Meanwhile,the effects of different CpxA modified strains on the expression levels of 15 known drug-resistant genes such as ompF,STM3031 and acrD were also determined.The results indicated that in all detected target genes associated with drug resistance,only the mRNA expression trends of ompW,acrD,spy,ycca,ppia,htpX and stm3031 genes were consistent among the three cpxA mutant strains.These results suggested that the regulation of various cpxA mutations for aminoglycoside(AGAs)and ?-lactam antibiotic resistance is dependent on CpxR key sites to regulate some common resistance genes resulting in increased drug resistance,which is not dependent on the efflux pump AcrAB-TolC.In this study,the clarification of regulation mechanism of CpxA on salmonella typhimurium multidrug resistance lay a foundation for related target antimicrobial drug research.