Effects Of The Two-component System BaeSR On The Antibiotic Resistance Of Salmonella Typhimurium

The multi-drug resistance of Salmonella is becoming more and more serious.It is an important means to study the resistance mechanism by obtaining a stable single antibiotic-resistant strain selected in vitro.Recent studies have shown that the two-component system(TCS)is closely related to bacterial antibiotics resistance.Therefore,S.typhimurium was selected in vitro to obtain the ciprofloxacin-resistant strain CR,and we take it as the study object,deleted mutants CR?baeSR were obtained by homologous recombination mediated by suicide plasmid pLP12.And we used chloramphenicol and arabinose-induced vmt toxicity gene for selecting.In addition,the complemental strains of CR C?baeSR were generated through transformation of a recombinant expression plasmid of pBAD33-Cm-rp4-baeSR into CR?baeSR.Then,the genetic stability of CR?baeSR and CR C?baeSR were tested,and the growth curves of three strains were determined.Furthermore,minimum inhibitory concentration(MIC)of 13 antibiotics against CR,CR?baeSR and CR C?baeSR strains were determined by broth microdilution method,biofilm formation and migration ability of three strains were also measured.Next,quantitative proteomic analysis of three strains was carried out by tandem mass tag(TMT)technique,and the results were verified by parallel reaction monitoring(PRM)targeted protein technology to discusse the relationship between two-component system BaeSR and antibiotic resistance in Salmonella typhimurium.The main research contents and results are as follows:1.Biological characteristics of CR,CR?baeSR and CR C?baeSR strains were detected.The growth curve,antibiotic sensitivity,the ability of biofilm formation and mobility of three strains were measured.The results indicated that compared with wild strains,the growth rate of CR?baeSR was slightly slower,but there was no significant difference(P>0.05);The MICs of CIP?ENR?SAR?CEF?GEN?AMK?APR?TET and DOX showed 2,2,2,4,2,2,2,2,2-fold decreased respectively compared with wild strains;The ability of biofilm formation(P<0.01)and migration(P<0.05)of CR?baeSR were decreased significantly.However,the related ability of baeSR gene has rebounded after complemented.This indicated that the two-component system BaeSR can affect its antibiotic resistance by regulating the ability of biofilm formation and motility in Salmonella typhimurium.2.The proteomics datas of TMT were processed and analyzed.There were 2422 proteins identified,2184 of which contained quantitative information.And the differential expressed proteins were screened at a 1.3-fold change threshold(fold change>1.3 or<0.67),P<0.05,the bioinformatics analysis was performed.The results showed that 186 differentially expressed proteins(68 were upregulated and 118 were downregulated)were screened in the CR ?baeSR/CR comparison group,and 562 differentially expressed proteins(227 were upregulated and 335 were downregulated)were screened in the CR C?baeSR/CR comparison group.In addition,the metabolic pathways of microbial metabolism,amino acid metabolism,carbon metabolism,sulfur metabolism and pyrimidine metabolism were significant enrichment.Indicating that the deletion of baeSR gene may lead to changes in various metabolic pathways,thereby affecting the expression of related proteins.3.Antibiotic resistance and TCS-related proteins were selected and target proteins were validated by PRM technology.According to the all annotation information of different proteins,19 resistance-related proteins were screened,including outer membrane proteins,ABC transporters,flagellin and biofilm formation-related proteins,which indicated that the deletion of baeSR gene in Salmonella typhimurium could affect the expression of other resistance-related proteins.17 TCS-related proteins were screened by classifying and counting the quantified proteins,suggesting that the two-component system BaeSR and other TCS may regulate each other,thus affecting the antibiotic resistance in Salmonella typhimurium.9 antibiotic resistance-related proteins were selected for PRM-targeted validation,the results were consistent with the trend of TMT,confirming the reliability of TMT quantitative proteomics results.In this study,baeSR gene deletion and complemental strains of Salmonella typhimurium were constructed and we investigated the effect of baeSR gene deletion on antibiotic resistance from the aspects of antibiotic sensitivity,biofilm formation ability,mobility and proteomics,so as to provide experimental basis for further study on the relationship between TCS and bacterial antibiotic resistance.