盐酸血竭素对金黄色葡萄球菌分选酶A活性影响研究

为探究盐酸血竭素(DP)抗金黄色葡萄球菌(S.aureus)分选酶A(SrtA)的活性及机制,本研究利用纤连蛋白黏附试验和细胞侵袭试验,验证DP通过抑制SrtA的活性影响S.aureus黏附于纤连蛋白和降低其对细胞的侵袭能力;利用荧光底物肽实验,测定DP对S.aureus SrtA的抑制作用;利用生物膜试验,观察DP对S.aureus形成生物被膜(BF)的影响能力;利用分子模拟和定点突变方法,分析验证DP对SrtA的活性中心影响作用。结果表明,DP分别在2μg/mL、4μg/mL、8μg/mL和16μg/mL时,可显著性抑制S.aureus SrtA蛋白的催化活性,且这一抑制作用呈浓度依赖性;DP可以通过影响SrtA的催化活性从而阻碍其表面蛋白锚定于宿主细胞表面,进而降低S.aureus侵袭细胞的能力;DP在8μg/mL和16μg/mL时可以显著降低S.aureus与纤连蛋白结合的能力(p<0.01);DP在2μg/mL、4μg/mL、8μg/mL和16μg/mL时均可以显著降低S.aureus形成BF的能力;DP可通过与SrtAΔN59中的P30和T105结合而封闭了其活性中心。综上所述,DP可作用于S.aureus毒力因子SrtA,通过抑制SrtA的活性达到减弱其毒力的效果。本研究将为DP抗S.aureus的作用机制提供参考依据。

A study on the effect of dracorhodinperochlorate on the activity of Staphylococcus aureus sorting enzyme A

To explore the activity and mechanism of dracorhodinperochlorate against Staphylococcus aureus(S.auruses)sorting enzyme A(SrtA),fibronectin adhesion and cell invasion tests were performed to verify the effect of dracorhodin hydrochloride(DP)on the adhesion of S.auruses to fibronectin and on reducing its invasion ability to cells through inhibiting SrtA activity.The inhibitory effect of DP on S.auruses SrtA was determined by fluorescence substrate peptide experiment.The biofilm test was used to examine the effect of DP on biofilm formation of S.aureus.Molecular simulation and site-directed mutagenesis methods were used to analyze and verify the effect of DP on the active center of SrtA in this study.Results showed that dracorhodinperochlorate significantly inhibited the catalytic activity of S.aureus SrtA protein at 2μg/mL,4μg/m L,8μg/mL and 16μg/mL,respectively,in a concentration-dependent manner.Dracorhodinperochlorate could inhibit the anchoring of surface proteins on the surface of host cells by affecting the catalytic activity of SrtA, thereby reducing the ability of S. aureus to invade cells. Draconitin hydrochloride at 8 μg/mL and 16 μg/mL significantly decreased the binding capacity of S. aureus to fibronectin(22.12% and 38.92%)(p<0.01). Draconitin hydrochloride at 2 μg/mL, 4 μg/mL, 8 μg/mL and 16 μg/mL significantly reduced the ability of S. aureus to form biofilm(p<0.01).Dragonin hydrochloride could occlude active center of SrtA by binding with P30 and T105 of SrtAΔN59. In conclusion, draconitin hydrochloride could act on virulence factor SrtA of S. aureus and inhibit the activity of SrtA toreduceits virulence. This study will provide a theoretical basis for the mechanism of draconitin hydrochloride against S. aureus.