| Biofilm is an important way for bacteria to resist drug stress,which is accompanied by the whole process of clinical infection.In natural state,more than 99%of bacteria grow in biofilm mode,which is beneficial to the survival and development of bacteria,and provides advantages for the evolution and variation of bacteria.Therefore,it is urgent to find a kind of natural active molecule which can inhibit the biofilm formation of Staphylococcus aureus and increase the sensitivity of antibiotics,so as to provide an effective solution for the increasingly serious bacterial drug resistance.1.In this study,SarA and its two mer proteins were expressed and purified by means of genetic engineering.The structural rationality of the protein was purified by gel retardation test,and the rabbit polyclonal antibodies were prepared.Based on the crystal structure of SarA protein,phylogenetic analysis,evolutionary pressure and structural characteristics were carried out in combination with GO,KEGG annotation and protein interaction network analysis.The results showed that the target proteins of 14.7kDa and 29.4kDa were purified,and the rabbit polyclonal antibody could specifically bind to them.SarA protein had many highly conserved sites,among which Lys85,Arg87,Asn88 and Asp91-Arg93 were the important active sites.In addition,SarA protein had rich interaction with bacterial colonization,biofilm formation and virulence expression.2.Based on the crystal structure analysis of SarA protein,the best binding pocket was determined,and the candidate inhibitors were screened from the constructed local natural active molecular data set,and their activities were verified in vitro,including the binding activities of SarA protein and candidate inhibitors,so as to determine that the candidate inhibitors have real and reliable target protein binding activities.The results showed that the best binding pocket was the cavity formed by His100,Ala138,Tyr142 and other amino acids.Andrographolide had a good binding performance in multi round docking,which was verified by molecular dynamics simulation.Andrographolide could inhibit the blocking effect of SarA protein on genomic DNA swimming,and could also quench the endogenous fluorescence of monomer SarA protein.The concentration of Andrographolide was 64μg/mL or above Andrographolide can significantly reduce the reactivity of monomer SarA protein with antibody,but not for dimer SarA protein,and circular dichroism and kinetic simulation further verify the above conclusion.In addition,Andrographolide also has the potential of binding with proteins such as AgrA and SasG.3.The minimum inhibitory concentration of Andrographolide on biofilm was determined by crystal violet staining,and the inhibition test on biofilm of 10 strains of S.aureus was carried out,then the expression difference of SarA protein in floating state and biofilm state was detected by Western-blot,and the expression of SarA protein after Andrographolide treatment was verified by qRT-PCR.The results showed that the minimum inhibitory concentration of biofilm was 32μg/mL,and the inhibitory activity was dose-dependent in the concentration range of 0～32μg/mL.The biofilm formation was significantly reduced when the concentration reached 64μg/mL,the content of SarA protein in biofilm state was significantly higher than that in floating state.Meanwhile,the content of dimer SarA protein decreased with the increase of Andrographolide concentration;qRT-PCR detected Andrographolide after lactone treatment,the transcriptional abundance of SarA gene only changed,which could inhibit the synthesis of extracellular DNA of intercellular adhesin.4.The whole transcripts of Andrographolide treated bacteria were sequenced by comparative transcriptomics,and the differentially expressed genes(DEGs)were obtained by statistical and bioinformatics methods,and the GO and KEGG enrichment analysis were carried out;the gene changes were verified by qRT-PCR;the potential antibiotic sensitization effect was detected by drug sensitivity test,and the morphology was observed by transmission electron microscope(TEM).The results showed that:113 genes with significantly different expression levels were detected,among which 54 genes were up-regulated,59 genes were down regulated,and the number of down-regulated genes was more than up-regulated,and they were mainly involved in the response to stress;qRT-PCR test and omics results showed consistent changes;it could effectively enhance the sensitivity of MRSA strains to Ampicillin,Penicillin G and Cefoxitin,and increase the inhibition zone fluoroquinolones also showed synergistic antibacterial effect to a certain extent,but no increase in the sensitivity of macrolides was observed.5.The β-lactamase BlaZ was obtained by prokaryotic expression system,and the binding of Andrographolide with BlaZ was verified by molecular simulation and fluorescence quenching test;the changes of Andrographolide with Penicillin G were observed by transmission electron microscope;the transcription levels of genes related to cell wall synthesis were detected by qRT-PCR.The results showed that:the target protein of 29.7kDa was obtained and belonged to type A enzyme;Andrographolide had a certain sensitizing effect on β-lactam antibiotics;when Penicillin was used alone,the cell wall of MRSA strain did not change,but when combined with Andrographolide,the cell wall was scattered,rough,even damaged and missing;the thickening of cell wall was mainly due to the increased synthesis of peptidoglycan,but the old peptidoglycan layer did not change The method was cleared by Atl in time,resulting in the uneven accumulation of cell wall and the thickening of cell wall.6.The whole protein mass spectrometry of Andrographolide treated bacteria was sequenced by TMT labeled quantitative proteomics,and the differentially expressed proteins(DEPs)were obtained by means of statistical and bioinformatics methods.The DEPs were analyzed by go and KEGG enrichment,and the DEPs were annotated comprehensively and in detail.The results showed that:a total of 344 DEPs were detected,of which 189 proteins were significantly down regulated and 155 proteins were up-regulated;similar to the results of transcriptomics,the number of down-regulated proteins was higher than that of up-regulated ones;SarA,a key subsidiary regulator,showed no difference in protein level;DEPs were involved in biofilm formation and quorum sensing and other biological processes.7.The RNA-Seq data were correlated with TMT labeled quantitative proteomics data to match the genes and proteins with differences at both levels;the above common differential genes and proteins were subdivided and hierarchical clustered by statistical methods,and the differences were annotated from different perspectives,including GO and KEGG enrichment analysis,and protein-protein interaction network analysis(PPI).The results showed that:there were two levels of consistent changes in 27,15 of which were up-regulated and 12 of which were down regulated;the difference was mainly involved in the infection caused by S.aureus,arginine metabolism,two-component regulatory system and biofilm formation.8.The datasets of GSE5466 and GSE139659,which were highly related to this study,were compared to obtain common differentially expressed genes,and GO and KEGG enrichment analysis and functional annotation were performed.The results are as follows:there were 119 common DEGs with GSE5466 data set,and 15 of them were related to biofilm formation;biofilm weak forming strain RN6390 and Andrographolide treated bacteria,sak,sspA,sspB,sspC genes had higher levels;after sarA gene knockout,there were 14 common DEGs with this study,and they were mainly located in the cell wall and peripheral membrane Andrographolide can enhance the sensitivity of β-lactams by activating the cell wall stress stimulon(CWSS)mechanism.In conclusion,SarA,the subsidiary regulatory factor of Staphylococcus aureus,was selected to obtain Andrographolide,a natural drug active molecule,and a series of studies on the inhibition of biofilm were carried out.Meanwhile,the potential antibiotic sensitization was verified and mechanism was explored.Then,the high throughput of transcribed and proteomics was used to obtain the different genes and proteins before and after Andrographolide treatment.The results of this study are as follows:The combination of histochemical association analysis and GEO database mining is used to reveal the internal mechanism of the inhibition of Staphylococcus aureus and antibiotic sensitization. |
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