红霉素通过IGPD干预木糖葡萄球菌生物被膜形成机制的研究

Study on the Mechanism of Erythromycin Inhibits Staphylococcus xylosus Biofilm Formation through Interacting with IGPD

为探究红霉素通过咪唑甘油磷酸酯脱水酶(IGPD)干预木糖葡萄球菌(Staphylococcus xylosus)生物被膜形成的机制,本试验采用肉汤微量稀释法测定红霉素对木糖葡萄球菌的最小抑菌浓度(MIC);采用结晶紫染色和扫描电镜试验检测红霉素对木糖葡萄球菌生物被膜的作用;利用Real-time PCR、酶活性测定、组氨酸含量测定等试验检测红霉素对IGPD的调控作用;采用分子对接和等离子共振试验验证红霉素与IGPD的直接相互作用。结果显示:红霉素MIC为1.6μg/mL,1/2 MIC(0.8μg/mL)的红霉素可以在不影响细菌生长的情况下极显著抑制生物被膜的形成(P<0.01);1/2 MIC(0.8μg/mL)的红霉素可以极显著抑制IGPD基因(hisB)的表达(P<0.01),同时显著降低IGPD的酶活性(P<0.05)和组氨酸含量(P<0.05);分子对接结果显示,红霉素可以与IGPD的氨基酸Arg88、Ser108、Leu40、Phe43、Glu162和Glu66位点结合;等离子共振试验结果显示,红霉素与IGPD可以直接结合。结果表明,红霉素可能通过调控IGPD及与其直接结合两方面作用,从而抑制木糖葡萄球菌生物被膜的形成。本试验结果为进一步明确红霉素干预木糖葡萄球菌生物被膜形成的机制及寻找干预木糖葡萄球菌生物被膜形成的药物靶点提供科学依据。

In order to explore the mechanism of erythromycin inhibition of Staphylococcus xylosus(S.xylosus)biofilm formation through interaction with imidazole glycerophosphate dehydratase(IGPD),this study utilized broth microdilution method to determine the minimum inhibitory concentration(MIC)of erythromycin against S.xylosus,crystal violet staining and scanning electron microscopy to test the effect of erythromycin on S.xylosus biofilm,real-time PCR,enzyme activity and histidine content assays to detect the regulatory effect of erythromycin on IGPD,and molecular docking and plasma resonance experiments to verify the direct interaction of erythromycin and IGPD.The results showed that the MIC of erythromycin was 1.6μg/mL,and 1/2 MIC(0.8μg/mL)of erythromycin significantly inhibited the formation of biofilm without affecting bacterial growth(P<0.01).1/2 MIC(0.8μg/mL)of erythromycin significantly inhibited the expression of IGPD gene(hisB)(P<0.01),and reduced the enzyme activity of IGPD(P<0.05)and the content of histidine(P<0.05)at the same time.In addition,molecular docking experiment revealed that erythromycin bound to the amino acid site Arg88,Ser108,Leu40,Phe43,Glu162 and Glu66 of IGPD.Plasma resonance experiment showed that erythromycin interacted with IGPD directly.Thus,erythromycin appears to inhibit the S.xylosus biofilm formation by regulating IGPD and combining with IGPD directly.This study lays the foundation for further elucidation on the mechanism and identification of drug targets of erythromycin inhibition of S.xylosus biofilm formation.