穿心莲内酯对耐甲氧西林金黄色葡萄球菌关键耐药蛋白PBP2a的抑制作用

Inhibition of key resistance protein PBP2a of methicillin-resistant Staphylococcus aureus by andrographolide

本试验旨在为探究穿心莲内酯(andrographolide, AP)对耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)关键耐药蛋白青霉素结合蛋白2a(penicillin binding protein 2a, PBP2a)的抑制作用。采用实时荧光定量PCR检测AP对PBP2a编码基因mecA的转录影响,通过在线软件SWISS-MODEL等对蛋白质结构进行分析,经PyMOL以及AutoDock Tools软件进行分子对接来探究AP与PBP2a结合的可能机制,运用动力学模拟来验证AP与PBP2a蛋白结合的可靠性,进一步原核表达PBP2a并制备其多克隆抗体,通过蛋白质免疫印迹检测AP作用下MRSA中PBP2a蛋白含量的变化。结果显示:64μg/mL的AP即可显著下调mecA的转录水平,AP与PBP2a蛋白的GLU170、GLU239和THR238之间可以形成稳定的氢键作用力;成功表达并纯化获得38 kDa的PBP2a蛋白转肽酶区,且制备出的多克隆抗体可以与PBP2a蛋白特异性结合;随着AP浓度的升高,PBP2a的表达量逐渐降低。总之,本研究分子模拟提示AP对PBP2a蛋白存在抑制潜力,试验结果表明AP可以通过抑制mecA的转录进而抑制PBP2a表达,从而揭示了AP使MRSA对β-内酰胺类抗生素增敏的可能机制。

This study was to explore the inhibitory effect of andrographolide(AP)on the critical drug-resistant and protein penicillin-binding protein 2a(PBP2a)of methicillin-resistant Staphylococcus aureus(MRSA).Here,the effect of AP on mecA gene transcription was detected by real-time fluorescence quantitative PCR.The protein structure was analyzed by the online software SWISS-MODEL.Pymol and AutoDock Tools software performed molecular docking to explore the potential mechanism of AP binding to PBP2a,and kinetic simulation was used to verify the reliability of AP binding to PBP2a protein.Then,prokaryotic expression of PBP2a was further performed,and its poly-clonal antibody was prepared.The change of PBP2a protein content in MRSA under the action of AP was detected by Western blot.The re-sults showed that AP significantly down-regulated the transcription level of mecA at 64μg/mL,and a stable hydrogen bond force was formed between AP and GLU170,GLU239,and THR238 of PBP2a protein.The transpeptidase domain of 38 kDa PBP2a protein was successfully expressed and purified,and the prepared polyclonal antibody specifically bound to the PBP2a protein.With the increase in AP concentration,the expression of PBP2a decreased gradually.In conclusion,the molecular simulation in this study suggested that AP had the potential to in-hibit PBP2a protein.The present results indicated that AP could inhibit the expression of PBP2a by inhibiting the transcription of mecA,thus revealing the possible mechanism of AP sensitizing MRSA toβ-lactam antibiotics.