| With the continuous emergence of multi-drug resistant strains of Salmonella,research on the mechanism of drug resistance has become the focus of the moment.It has been confirmed that two-component signal transduction system and multiple drug resistance efflux pump play an important role in the mechanism of Salmonella resistance.In addition,the presence of acrB may mask the function of other efflux pumps.Therefore,in this study,a homologous recombination system mediated by suicide plasmid p LP12 was used to construct the acrB gene-deficient strains of Salmonella typhimurium induced ciprofloxacin resistant strains CR and CR?bae SR in vitro,and compared the drug sensitivity and biological function of the strains before and after the deletions.Transcriptomics technology and EMSA test were used to find the potential target genes of Bae R,and the phosphorylation quantitative proteomics technology was used to screen the differentially expressed phosphorylated proteins of the tested strains at the post-translational modification level of the protein.The aim is to elucidate the regulatory mechanism of Bae SR and Acr B resistance in S.typhimurium at different levels.The main research contents and results are as follows: 1.Construction and biological characteristics analysis of the acrB gene deletion strains of S.typhimuriumThe p LP12 plasmid combined with the forward and reverse screening technology was used to construct acrB gene deletion strains of CR,CR ? bae SR.Chloramphenicol resistance was the forward screening condition,and the vmt gene(with arabinose-induced suicide lethal gene)was the reverse screening condition,and further verified by PCR and sequencing.The results showed that CR?acrB and CR?bae SR?acrB were successfully screened.The MIC,growth characteristics,biofilm formation ability,and motility of the tested strains were tested respectively.The results showed that compared with the standard strain CR,the MIC of CR?acrB against ceftiofur,azithromycin decreased by 87.5%,amoxicillin,enrofloxacin,ciprofloxacin decreased by 75%,and the MIC of the remaining 7 drugs(ampicillin,amikacin,ampramycin,tetracycline,doxycycline,florfenicol,chloramphenicol)decreased by 50%;compared with CR?acrB,CR?bae SR?acrB did not change the MIC of penicillins(ampicillin and amoxicillin),the MIC of flufenicol and chloramphenicol decreased by 87.5% or more,and the other drugs decreased by 50%;there was no significant difference in the growth rate of the strains before and after deletion,indicating that the deletion of bae SR and acrB genes did not affect the growth rate of S.typhimurium;compared with CR,the biofilm formation ability of CR?acrB and CR?bae SR?acrB was significantly weakened(P<0.01),and CR?bae SR?acrB was more obvious than CR?acrB;measurement of the diameter of the tested strains' mycelium revealed that their motility change were consistent with the biofilm change trend.This indicates that Acr B and Bae SR can coordinately regulate the multidrug resistance of Salmonella by regulating bacterial biofilm formation and motility.2.RNA-Seq screening bae SR,acrB differential genes before and after gene deletion and identification of Bae R downstream targetsThe transcriptomics datas of CR,CR?acrB,CR?bae SR?acrB were analyzed.With |log2(Fold Change)|>1 and qvalue<0.005 as the screening conditions,a total of 1320 differentially expressed genes were screened in the CR?acrB/CR comparison group,894 were down-regulated and 426 were up-regulated;a total of 1377 differentially expressed genes were screened in the CR ? bae SR ? acrB/CR comparison group,972 were down-regulated and 405 were up-regulated.Bioinformatics analysis of the differential genes showed that the differential genes were mainly enriched in pathways such as metabolic pathway,ABC transport system,two-component signal transduction system,flagellar assembly,and ?-lactam resistance.According to gene annotation and literature reports,a total of 16 drug resistance-related differentially expressed genes were screened,involving efflux pump,biofilm,two-component system,chemotaxis and so on.Random screening of 7 differentially expressed genes was basically consistent with the q RT-PCR detection results,which confirmed the reliability of RNA-Seq analysis results.The Bae R protein was expressed in prokaryotic using p Cold I expression vector,purified by Ni-His resin column affinity chromatography and Western blot verified.The results showed that Bae R protein could be expressed in the form of inclusion body or in the supernatant.After purification,it was verified that the band of interest clearly appeared at 28 KD,indicating that the Bae R protein was successfully expressed and active.csg F,stm1545,fli A,mdt C,pmrf and stm3031 were selected as target genes of Bae R and the gel retardation test was carried out according to the promoter region of the DNA fragment containing biotin labelled.The results showed that Bae R could directly regulate the expression of mdt C and pmr F and thus regulate the resistance of Salmonella.3.Phosphorylation quantitative proteomics screening of differentially expressed proteins before and after bae SR and acrB gene deletionThe relative quantification of post-translational modification in the tested strains was carried out by the combination of protein quantitative technique and modification enrichment technique to screen the drug resistance related differential modified proteins.The results showed that a total of 213 modified proteins were identified in the four tested strains,containing a total of 363 modified sites.In the comparison group,CR?bae SR/CR was used to identify 80 differential expression proteins(34 were down-regulated,46 were up-regulated);CR?acrB/CR was used to identify 128 differential expression proteins(103 were down-regulated,25 were up-regulated);CR?bae SR?acrB/CR was used to identify 96 differential expression proteins(66 were down-regulated,30 were up-regulated).Bioinformatics analysis showed that protein phosphorylation was involved in the transport,transcription,metabolic pathways,virulence and drug resistance of bacterial cells.Functional classification of differential proteins identified a total of 21 proteins associated with bacterial resistance and 4 proteins associated with virulence.According to the functional classification of differential proteins,21 kinds of proteins related to bacterial resistance and 4 kinds of proteins related to virulence were identified.To sum up,in this study,acrB gene deletion strains CR?acrB and CR?bae SR?acrB were successfully constructed by homologous recombination system mediated by suicide plasmid plp12.The drug sensitivity results showed that the strains with bae SR and acrB gene deletion were more sensitive to a variety of antibiotics.Combined with the changes of biological characteristics,transcriptome and EMSA,we found that bae SR and acrB can regulate the multiple drug resistance pathways of S.typhimurium,such as biofilm formation ability,motility,efflux pump expression,two-component system and bacterial chemotaxis,etc.Meanwhile,Bae R can also directly regulate the expression of mdtc and pmr F,so as to participate in the regulation of the drug resistance of S.typhimurium.In addition,the results of quantitative proteomics of phosphorylation showed that phosphorylation was involved in the regulation of drug resistance of S.typhimurium at the level of post-translational modification. |
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