| Shigella is the causative agent of bacillary dysentery,with a wide range of prevalence,high infection rate and serious harm,which is one of the important global public health challenges.With the widespread use of antibiotics in clinical practice,the resistance burden of Shigella has become increasingly prominent,which has become the main reason for the failure of drug treatment for Shigella infection.In recent years,Shigella sonnei has become the main epidemic strain and has high resistance to antibiotics,such as ampicillin,cephalosporins and ciprofloxacin.We conducted a preliminary study on the regulation of cfa gene on Shigella resistance,including induced a sensitive Shigella strain(WT strain)to become resistant to ampicillin.And homologous recombination technology was used to construct the cfa gene deletion strain(Res-Δcfa strain)of drug-resistant strain(Res strain).Compared with the WT strain,the m RNA expression of the cfa gene in the Res strain was increased by 3 times,suggesting that the cfa gene may regulate the resistance of Shigella sonnei to ampicillin.In order to further clarify the molecular mechanism of the cfa gene regulating the resistance of Shigella sonnei,this study used the microbroth dilution methods to compare the susceptibility of Res and Res-Δcfa strains to ampicillin.The differentially expressed proteins and metabolites of the two strains were screened by proteomic and metabolomic techniques.To explore the regulatory pathway of cfa gene against Shigella resistance,to provide new ideas and insights for solving the problem of bacterial resistance and developing new antibiotics.ObjectiveTo Explore the molecular regulation mechanism of cfa gene resistance to Shigella sonnei,screen and verify the differential proteins and differential metabolites related to drug resistance through proteomics and metabolomics technology,and provide drugs targets and theoretical basis for the prevention and treatment of drugresistant Shigella infection.Methods1.PCR and sequencing were used to verify the cfa gene in the Res and Res-Δcfa strains.2.The minimum inhibitory concentration(MIC)of the Res and Res-Δcfa strains to ampicillin were detected by the microbroth dilution methods.3.Quantitative Real-time PCR(qRT-PCR)was used to detect the m RNA expression of cfa gene among Res,Res-Δcfa,multi-drug resistant strain N1 and N4.4.Construct the cfa gene overexpression vector p ET28a-cfa and transfer it into Res and Res-Δcfa strains.The MIC of Res-p ET28 a,Res-Δcfa-p ET28 a,Res-p ET28 acfa and Res-Δcfa-p ET28a-cfa strains to ampicillin antibiotics were detected by microbroth dilution method.5.The strains Res and Res-Δcfa were compared by proteomics,and the differentially expressed proteins of the two groups of strains were screened by bioinformatics analysis,and the m RNA transcription level of the proteins that may be related to the resistance of Shigella sonnei were verified.6.Metabolomics analysis was performed on the Res and Res-Δcfa strains,and the differentially expressed metabolites between the two strains and the metabolites that may be related to the resistance of Shigella sonnei were screened out.Transcriptional level validation of genes on differential metabolite enrichment pathways.7.Analyze the biological characteristics of strains Res and Res-Δcfa,and detect the growth status,biofilm formation ability and virulence of the two strains by bacterial growth curve determination,biofilm formation experiment and Cell Counting Kit-8(CCK-8)experiment.Results1.Both PCR identification and sequencing verification indicated that the cfa gene of Res-Δcfa strain was deleted.2.The MICs of the Res and Res-Δcfa strains to ampicillin were 128 μg/m L and 2μg/m L,respectively.Res-Δcfa was 64-fold more sensitive to ampicillin compared to the Res strain.3.The results of qRT-PCR showed that,compared with Res,the expression level of cfa gene in Res-Δcfa decreased(P < 0.001).In addition,the expression levels of cfa genes in the multi-drug resistant strains N1 and N4 were comparable to or higher than those of the Res strain,supporting the important role of cfa genes in bacterial drug resistance.4.The cfa gene overexpression vector p ET28a-cfa was successfully constructed,and p ET28a-cfa was transformed into the Res-Δcfa strain,and its MIC to ampicillin increased from 2 μg/m L to 4 μg/m L.5.Proteomic analysis showed that compared with Res,Res-Δcfa strain was identified 81 up-regulated proteins and 211 down-regulated proteins.The Gene Ontology(GO)functional pathway with significantly enriched differential proteins was the lyase activity pathway,and the significantly enriched Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway was sulfur metabolism and microbial metabolism in different environments.Deletion of the cfa gene results in L-serine dehydratase(Tdc G),NAD-dependent dihydropyrimidine dehydrogenase subunit(Pre A),ferrous transporter A(Feo A),protein(Mtf A),putrescine transporter(Pot E),and DNA-binding response regulator protein(Uvr Y),adenylate sulfate transferase subunit 2(Cys D),spermidine/putrescine ABC transporter substrate-binding protein(Pot F),chaperone regulatory protein(Cbp M),and other proteins changed significantly.6.qRT-PCR was used to detect the m RNA level of adi C,btu B,cys N,cys H,cys I,idn D,met E,mtf A,and pot E,and found that there was significant difference in the transcription level of the above genes between Res-Δcfa and Res strains(P < 0.05)7.Metabolomic analysis showed that compared with Res,Res-Δcfa strain screened out 149 differential metabolites(99 up-regulated,50 down-regulated)in positive ion mode;122 differential metabolites were screened out in negative ion mode(39 up-regulated,83 down-regulated).The differential metabolites of the two strains were significantly enriched in the cysteine and methionine metabolic pathways(P < 0.05).In the positive ion mode,the differential metabolites between Res-Δcfa and Res strains are mainly manifested in S-adenosylmethionine(SAM),O-acetyl-Lhomoserine,S-adenosylhomocysteine(SAH),O-phospho-L-serine;while in negative ion mode,the two groups of differential metabolites mainly showed methionine,Lhomocysteine and L-aspartic acid.8.The expression levels of genes in the cysteine and methionine metabolism pathways of Res-Δcfa and Res strains were verified by qRT-PCR.The results showed that compared with Res,the serine acetyltransferase on this pathway of Res-Δcfa strains Genes(cys E),cysteine synthase(cys K),S-ribose homocysteine lyase(lux S)upregulated,methionine adenosyltransferase(met K)and the homocysteine methyltransferase gene(met E)was down-regulated,and the difference was statistically significant(P < 0.05).9.Under no antibiotic pressure,Res-Δcfa grows faster than Res strain and has stronger biofilm formation ability,but the culture supernatant of the two strains has no significant difference in Vero cytotoxicity.Conclusion1.The cfa gene regulates the resistance of Shigella sonnei to ampicillin.After knocking out the cfa gene,Res-Δcfa were more sensitivity to ampicillin.When the Res-Δcfa strain expressed the cfa gene again,its sensitivity to ampicillin decreased.2.Proteomics and metabolomics analysis found that the expression of cysteine and methionine pathway-related proteins(Met E and Met K)and metabolites(SAM and SAH)may play role in the molecular mechanism of ampicillin resistance in Shigella sonnei regulated by cfa gene.3.The cfa gene regulated the growth and other life activities of Shigella sonnei.Compared with the drug-resistant strain Res,the Res-Δcfa knockout strain has higher biofilm formation ability. |
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