Inhibition Of Methicillin-resistant Staphylococcus Aureus Major Resistance Protein PBP2a By Andrographolide

Penicillin-binding proteins(PBPs)are key membrane proteins in bacterial cell wall formation.β-lactam antibiotics can bind PBPs,thereby affecting the biosynthesis of cell wall peptidoglycans and acting as bactericides.Methicillin-resistant Staphylococcus aureus(MRSA),a typical drugresistant bacterium,produces PBP2a with low affinity for antibiotics leading to broad-spectrum resistance.pBP2a is considered a potential target protein for drug development,and plant-derived natural small molecule andrographolide(AP)is a class of multi-target terpenoids that can affect the susceptibility of S.aureus to drugs,so inhibition of PBP2a by AP to inhibit the activity of PBP2a and restore the sensitivity of MRSA to β-lactam antibiotics or reduce the drug resistance of MRSA is of great research importance.1.Bioinformatics was used to investigate the structure and conservativeness of the PBP2a protein,and the ability of AP to interact with the PBP2a protein was predicted using molecular docking analysis,which showed that AP can form hydrogen bonds through interaction with residues such as PRO258,AL277 and TYR373 of the PBP2a protein,and inter-alkyl molecular forces,while,at the same time,will form a complex under π-alkyl stacking forces to form complexes,the binding of these amino acid residues to AP may compromise its biological function,and strong binding to TYR373,a highly conserved functional region of the protein.Finally,molecular dynamics simulations were used to verify that AP can form stable complexes with the PBP2a protein,indicating that AP has some potential to inhibit the PBP2a protease.2.The effect of AP on the transcription level of mecA gene was examined by real-time fluorescence quantitative PCR,and the results showed that AP could significantly down-regulate the transcription level of mecA gene.The recombinant protein PBP2a and the polyclonal antibody of this recombinant protein were obtained by prokaryotic expression and polyclonal antibody biotechnology,and the expression level of PBP2a protein in MRSA strains under the effect of different concentrations of AP was detected by using immunoblotting method.The results showed that the prepared polyclonal antibody to the recombinant protein PBP2a could react specifically with the PBP2a protein in MRSA bacteria,and the results showed that the expression of PBP2a gradually decreased with the increase of AP concentration.3.The effect of AP on the transcription level of crtM gene was examined by methanol extraction of carotenoids in MRSA strains and real-time fluorescence quantitative PCR.The results showed that there was a trace inhibition of carotenoids by AP,and the real-time fluorescence quantitative PCR results showed that AP could down-regulate the transcription level of this gene,so AP could inhibit the synthesis of intermediate intermediates of grape xanthin by inhibiting the catalase CrtM The results of real-time fluorescence quantitative PCR showed that AP could down-regulate the transcript level of this gene,so AP could inhibit the synthesis of vitexin by inhibiting the synthesis of CrtM,the catalase of vitexin synthesis intermediate.In summary,andrographolide inhibits the key MRSA resistance protein PBP2a,andrographolide can bind to PBP2a protein to form a stable complex,inhibit the transcription level of mecA gene,reduce the expression of PBP2a protein,and affect the localization of PBP2a on the cell membrane to influence its function by inhibiting the synthesis of carotenoids.Thus,the sensitivity of MRS A to β-lactam antibiotics was improved.