| Methicillin-resistant Staphylococcus aureus(MRSA)is one of the most common pathogens in nosocomial infections,and due to the widespread and unreasonable use of antibiotics,the problem of MRSA resistance has become increasingly serious.Previous studies have shown that the formation of active efflux systems and biofilms of MRSA is the main cause of multi-drug resistance.Bacterial resistance caused by the formation of active efflux systems and biofilms is associated with resistance-related proteins.The preliminary results of our laboratory also proved that the genistein Chinese herbal compound can significantly inhibit the expression of MRSA protein,and it is speculated that its inhibition on MRSA is related to protein,but after the effect of genistein on MRSA,the bacterial protein The change is still unclear.Therefore,genistein was used as experimental material to detect the change of bacterial protein expression after MRSA by iTRAQ technology in this study.And through bioinformatics analysis methods,systematically analyze the differences between the significant proteins in terms of molecular function,biological processes,location of the cells,and pathways involved;The mechanism of action of drug resistance-related proteins in mediating bacterial resistance was also discussed.The specific findings are as follows:(1)The change of the expression of bacterial protein after MRSA was detected by iTRAQ technique.The experimental results showed that a total of 1312 proteins were detected in the sample.Among them,there were 129 differentially significant proteins,including 60 proteins with up-regulated expression and 69 proteins with down-regulated expression.(2)The mRNA expression level of the differentially significant protein was detected by Real-time PCR to verify the accuracy of the iTRAQ test results.The results showed that compared with the control group,the mRNA expression levels of the down-regulated proteins PstB and PstC were significantly decreased,and the gene expression levels were decreased by 51.6% and 52.1%,respectively.The expression levels of the up-regulated proteins SecY,Mip,and RecT were significantly increased.The gene expression levels increased by 77.2%,87.5%,and 90.1%,respectively.The expression level of the gene is consistent with the trend of protein expression level,indicating that the iTRAQ test results of this study are accurate and can be used for subsequent bioinformatics analysis.(3)Bioinformatics analysis was performed on the difference of significant protein expression after MRSA by genistein by GO,KEGG and String methods.1)GO analysis showed that 129 significant differential proteins were mainly involved in 10 biological processes.According to the proportion of genes,the main involved were metabolic processes(80%),cellular processes(65%),and single organisms processes(58%)and so on.Mainly distributed in cells(46%),cell components(44%),cell membrane(22%),cell membrane components(18%),macromolecular complex(16%)and other locations.Mainly perform 7 kinds of molecular functions,according to the ratio of catalytic activity(63%),binding function(44%),transport activity(10%),structural molecular activity(6%),molecular transduction activity(3%))2)KEGG pathway database analysis showed that 129 differentially significant proteins mainly include four major pathways,namely metabolic pathway,genetic information processing pathway,environmental information processing pathway and some unknown pathways.Among them,the metabolic pathway contains 50 differential proteins,the genetic information processing pathway contains 17 differential proteins,and the environmental information processing pathway contains 17 differential proteins.3)String analysis showed that there were direct and indirect interactions between 129 differentially significant proteins.Some proteins are densely connected and some are loosely connected.Among them,secY,rpsE,isaB,and PstB proteins have an interactive relationship with other proteins ?5,which is the central node of protein interaction network,and plays an important role in protein transport,ribosome synthesis,cellular immunity,and bacterial resistance.(4)Screening of drug resistance-associated proteins in significantly different proteins by bioinformatics analysis.The results showed that there were about 14 proteins related to bacterial resistance.Among them,PstB mainly mediates bacterial resistance through active efflux system.PstS mediates bacterial resistance mainly by promoting the formation of bacterial biofilm.(5)The mechanism of PstB and PstS protein-mediated bacterial resistance was verified by accumulation kinetics experiment,crystal violet semi-quantitative method and qRT-PCR method.1)The results of accumulation kinetics showed that the amount of ciprofloxacin accumulated in the cells was significantly higher than that of the control group after verapamil treatment of MRSA41577 cells.Among them,after 10 min of verapamil-treated with 100 ?g/ml,the accumulation of ciprofloxacin increased by 32% compared with the blank control group(P<0.01).This indicates that verapamil can inhibit the efflux of ciprofloxacin by MRSA efflux pump protein.Since PstB is a constituent protein of the efflux pump,reversal of bacterial resistance by verapamil may be related to inhibition of PstB protein expression.Real-time PCR results showed that the expression of pstB gene was significantly decreased after treatment with MRSA41577 for 16 h at 100 ?g/ml verapamil.Compared with the control group,the expression level of pstB gene was decreased by 89%(P<0.01).It is indicated that verapamil reverses MRSA resistance by inhibiting the expression level of pstB mRNA.2)Semi-quantitative experiments of crystal violet showed that 100 ?g/ml verapamil had a certain inhibitory effect on the formation of MRSA41577 biofilm and the mature biofilm.Compared with the control group,the number of cells was reduced by about 25%(P<0.05).The inhibitory effect may be to inhibit the expression of pstS gene by verapamil,thereby inhibiting the formation of biofilm.Real-time PCR results showed that the expression of pstS gene was significantly decreased after treatment with MRSA41577 for 16 h at 100 ?g/ml verapamil.The expression level of the pstS gene was reduced by 90%(P<0.01)compared to the control group.Indicating that verapamil is by inhibiting the pstS mRNA. |
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