| BackgroundDrug resistance of Escherichia coli has become a worldwide problem.Many factors can influence the emergence of drug resistance,such as the characteristics of strains and the application of antibiotics.Antibiotics are the main means of prevention and treatment of Escherichia coli infection in clinic,but the irregular use of antibiotics leads to more and more serious drug resistance of pathogenic.In recent years,the problem of multi-drug resistance of Escherichia coli has become more and more serious.At the same time,drug resistance of animal-derived Escherichia coli has become more serious too.The production of extended spectrum ?-lactamases is an important mechanism of drug resistance Escherichia coli,which brings great difficulties for clinical treatment.Extended spectrum ?-lactamases producing Escherichia coli is usually highly resistant to cephalosporins such as ceftazidime and cefotaxime,but more sensitive to ?-lactamase inhibitors.However,in clinical practice,more than 10% of Escherichia coli producing extended spectrum ?-lactamases has exhibited resistance to ?-lactamase inhibitors,which leads to the increasing of infection-related mortality Directly.These results suggest that there may be other molecular mechanisms involved in the development of drug resistance in Escherichia coli producing extended spectrum ?-lactamases.Therefore,it is necessary to study the related mechanism of extended spectrum ?-lactamases production in drug-resistant Escherichia coli.Objective1.We screen the differently expressed proteins and metabolites between extended spectrum ?-lactamases producing Escherichia coli and the sensitive Escherichia coli with the methods of proteomics and non-target metabolomics,in order to reveal the differential protein expression profile and metabolic trend of them,and provide scientific basis for the research on the drug resistance of the bacteria at the level of protein and metabolite.2.We further analyze the different expressed proteins and metabolites results through bioinformatic methods in order to find the significantly related metabolic pathways of extended spectrum ?-lactamases producing Escherichia coli,which can be the foundation for further key gene selection.3.Find the key genes which were significantly related to the production of extended spectrum ?-lactamases in drug-resistant Escherichia coli.4.With the methods of gene knockout,construction of overexpressed plasmids and other techniques for the experimental strains,and the changes in the production of extended spectrum ?-lactamases production were monitored,in order to reveal the influence of the different expressed genes on the production of extended spectrum ?-lactamases in drug-resistant Escherichia coli,and provide a new direction for the treatment and antibiotic development of Escherichia coli producing extended spectrum ?-lactamases.Method1.We used the method of proteomics to screen the different expressed proteins betweem extended spectrum ?-lactamases producing Escherichia coli and the sensitive Escherichia coli.10 cases of extended spectrum ?-lactamases producing Escherichia coli and 10 cases of sensitive Escherichia coli were selected.The experimental group and the control group were divided into 3 parts after sample equivalent mixing,the differential expressed protein between the two groups were screened by tandem mass label proteomics.Basic data processing,hierarchical clustering analysis,KEGG Pathway analysis,and differential protein network construction analysis were performed for the results.2.We used the method of untarget metabolites to screen the differential metabolites between the two groups.10 cases of extended spectrum ?-lactamases producing Escherichia coli and sensitive Escherichia coli were selected,the differential metabolites between the two groups were screened and the results were analyzed by principal component analysis,orthogonal partial least squares discriminant analysis,orthogonal partial least squares discriminant substitution test,differential metabolite screening,and differential metabolite and related metabolic pathway attribution analysis.3.Bioinformatic methods were used to analyze the results of different expressed proteins and differential metabolites between the two groups.First,Spearman correlation coefficient was used to analyze the correlation of differential proteins and differential metabolites,and then KEGG database was used to analyze the correlation pathway of differential metabolites and differential proteins,then a correlation network between different expressed proteins and metabolites was constructed.By enrichment analysis,the metabolic pathways significantly associated with the production of extended spectrum ?-lactamases by drug-resistant Escherichia coli were obtained.4.The 2-deoxyadenosine monohydrate,2,6-dihydroxypurine and xanthine in the significantly enriched purine metabolic pathway were further quantitatively verified by the target metabolomics method.At the same time,factors such as MS2 score,P value,VIP and metabolite-protein interaction were considered comprehensively,the reliability of our analysis method was evaluated by combining the results of target proteomics analysis with the results of non-target metabolomics data.5.Whole gene frame of the experimental Escherichia coli were sequenced respectively.In combination with the significantly enriched pathways in the correlation analysis results,the differential genes were screened out after comprehensive analysis,and then they were verified by real-time fluorescent quantitative PCR technology.The results showed that GLO1 gene in pyruvate metabolic pathway was highly expressed.Construct GOL1 gene knockout and overexpression strains,then the production of ?-lactamases were examined by Elisa respectively.Results1.Tandem quality label proteomics analysis revealed 1553 differentially expressed proteins between the two groups.And the enrichment results showed that the differentially expressed proteins were functionally enrichment in 53 GO items(p < 0.05).Among them,29 were related to biological processes,7 were related to cellular components,and 10 were related to molecular functions.2.A total of 1165 differential metabolites of the two groups were screened by untargeted metabolites analysis,among which 350 were up-regulated and 815 were down-regulated.Pathway analysis based on differential metabolites revealed 82 different metabolic pathways between the two groups.In the positive ion mode,the significantly different metabolic pathways were purine metabolic pathway,arginine and proline metabolic pathway,pyruvate pathway,nicotinate and nicotinamide pathway,vitamin B6 pathway,streptomycin biosynthesis pathway,etc.The significantly different metabolic pathways in the anion mode were the biosynthesis of unsaturated fatty acids,the nicotinate and niacinamide pathways,the biosynthesis of pantothenate and coenzyme A,the pyrimidine metabolic pathways,glycerolipids,glycerides,and glycerides,etc.3.The correlation analysis of metabolomics and proteomics data showed that 18 pathways may be significantly correlated with production of extended spectrum ?-lactamases for Escherichia coli.The different pathways obtained by correlation analysis included purine metabolic pathway,arginine and proline metabolic pathway,pyruvate pathway,nicotinate and nicotinamide pathway,unsaturated fatty acid biosynthesis pathway,pantothenate and Co A biosynthesis pathway,pyrimidine metabolic pathway,etc.4.There were three metabolite candidates in the purine metabolic pathway,which were 2-deoxyadenosine monohydrate,2,6-dihydroxypurine and xanthine,and they were further quantitative verified by super-high performance liquid chromatography multi-reaction monitoring mass spectrometry,the coincidence of the MRM analysis results and non-target metabolomics data results showed the reliability of our analysis method.5.The results of genome-wide framework and the above 18 differential metabolic pathways were comprehensively analyzed to screen out the different genes,which included add,pun A,gua D,apa H,adk,prd A,spe G,Ace deacetylase,PRODH,cod A,hch A,frd A,GLO1,ppn K,ilv E,pan decarboxylase,VNN,tdk,deo A,tmk.The expression of the candidate m RNA in standard strain ATCC25922,sensitive Escherichia coli and extended spectrum ?-lactamases producing Escherichia coli were analyzed by real-time fluorescence quantitative PCR.The results showed that there was no difference in the expression of these m RNA in standard bacteria ATCC25922 and sensitive Escherichia coli,and the relative expression levels of spe G,acetylputrescine deacetylase,GLO1 and Pantothenoylcysteine Decarboxylase were significantly increased in Escherichia coli producing extended spectrum ?-lactamases,while the expression levels of the other genes were decreased.6.Constructed GOL1 gene knockout and overexpression strains,and then the effects of GLO1 gene on the ?-lactamase subtypes were analyzed by Elisa.A total of 10 subtypes were analyzed,including ?-lactamase BES,CTX-M1,CTX-M2,OXA1,OXA2,OXA10,PER,SHV,TEM,and VEB.The results showed that GLO1 gene expression level only affected PER type of ?-lactamase,but had no significant effect on other types of ?-lactamase.The expression of PER ?-lactamase significantly increased after GLO1 overexpression plasmids were transfected into standard strain ATCC25922 and sensitive Escherichia coli.The expression of PER ?-lactamase was significantly reduced in Escherichia coli producing extended spectrum ?-lactamases after the GLO1 was knocked out,and this reduction could be reversed by the transfection of GLO1 overexpressed plasmid.These results showed that the changes of GLO1 gene expression level were significantly correlated with the expression level of PER ?-lactamase in Escherichia coli.Conclusion1.The protein profile of Escherichia coli producing extended spectrum ?-lactamases was significantly changed,and the overall trend of down-regulation was observed.Differential expressed proteins are involved in most aspects related to biological processes,cell components and molecular functions.2.The metabolites of Escherichia coli producing extended spectrum ?-lactamases were significantly changed,and the metabolites were mainly down-regulated.3.GLO1 was significantly associated with the drug resistance of Escherichia coli,and its effect on drug resistance was mediated by increasing PER ?-lactamase production. |
PDF Full Text Request