甘胆口服液对副溶血弧菌的抑菌作用及转录组分析

Inhibition effect and transcriptome analysis of Gan Dan oral liquid against Vibrio parahaemolyticus

摘要:

目的 探究甘胆口服液(Gan Dan oral liquid, GD)对副溶血弧菌的体外抑菌活性，并从转录组水平解析GD抗副溶血弧菌的分子机制。方法 基于最小抑菌浓度(minimum inhibitory concentration, MIC)、最小杀菌浓度(minimum bactericidal concentration, MBC)和抑菌动态生长曲线，评价GD对副溶血弧菌的体外抑菌活性；通过扫描电镜(scanning electron microscopy, SEM)和透射电镜(transmission electron microscopy, TEM)分析GD处理对副溶血弧菌细胞结构的影响；利用转录组测序及生物信息学技术分析1/4MIC浓度的GD处理对副溶血弧菌转录组的影响，并采用逆转录实时定量聚合酶链式反应(real-time quantitative reverse transcription PCR, RT-qPCR)进行验证。结果 GD对副溶血弧菌具有较好体外抑菌活性，其MIC和MBC值分别为7.6 mg/mL和 15.2 mg/mL。1/4MIC浓度的GD处理破坏了副溶血弧菌细胞壁结构完整性，影响了细胞膜的通透性，导致胞内大分子物质外漏。转录组分析结果表明，GD处理显著改变了副溶血弧菌的转录组，造成1 074个基因显著上调，以及1 179个基因显著下调。其中，下调基因主要富集在肌苷酸、核糖核苷合成代谢以及柠檬酸循环等通路，上调基因主要涉及转录因子活性、细胞壁合成代谢等通路。结论 GD可能通过破坏菌体细胞壁和细胞膜结构的完整性，抑制细菌的能量代谢和生物合成等功能，进而对副溶血弧菌产生抑菌作用。

Abstract:

Objective To investigate the in vitro inhibitory activity of Gan Dan oral liquid (GD) against Vibrio parahaemolyticus and decipher the inhibition mechanism at the transcriptome level.Methods The in vitro inhibitory activity of GD against V. parahaemolyticus was evaluated based on the minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and growth curve. Scanning electron microscopy and transmission electron microscopy were employed to analyze the effect of GD on the cellular structure of V. parahaemolyticus. Transcriptome sequencing coupled with bioinformatics methods were employed to investigate the effect of GD at 1/4MIC on the transcriptome of V. parahaemolyticus, and the obtained results were examined by real-time quantitative reverse transcription PCR (RT-qPCR).Results GD exhibited high in vitro inhibitory activity against V. parahaemolyticus, with the MIC and MBC of 7.6 mg/mL and 15.2 mg/mL, respectively. The GD treatment at a concentration of 1/4MIC disrupted the cell wall integrity and increased cell membrane permeability of the pathogen, and leading to the leakage of intracellular macromolecules. The results of transcriptome sequencing showed that GD treatment significantly altered the transcriptome profile of V. parahaemolyticus, resulting in significant upregulation of 1 074 genes and significant downregulation of 1 179 genes. The downregulated genes were mainly enriched in pathways related to the synthesis of inosinate and ribonucleoside, whereas the upregulated genes were primarily categorized into pathways associated with transcription factor activity and cell wall synthesis.Conclusion GD may inhibit V. parahaemolyticus by disrupting cell structure and inhibiting cellular energy metabolism and biosynthesis.