大蒜素对肺炎克雷伯氏菌的抑制作用及其机制分析

Inhibitory effect and mechanism of allicin on Klebsiella pneumoniae

摘要:

背景 肺炎克雷伯氏菌(Klebsiella pneumoniae)是一种常见致病菌，然而其临床用药较局限，筛选开发新的药物对于其防治尤为重要。大蒜素具有多重药理功能，关于大蒜素抑菌有部分研究但不够深入，并且大蒜素影响肺炎克雷伯氏菌毒力研究鲜有报道。目的 探究大蒜素对肺炎克雷伯氏菌生长及毒力因子的影响及其机制。方法 在含有不同浓度(25 μg/mL和50 μg/mL)大蒜素的液体培养基中，检测细菌生长曲线与存活率，使用微量肉汤稀释法检测大蒜素的最小抑菌浓度(minimum inhibitory concentration, MIC)与最小杀菌浓度(minimum bactericidal concentration, MBC)；显微镜观察细菌形态，并检测膜电势和膜通透性；表型分析生物膜、荚膜、菌毛的形成能力；利用RT-qPCR分析毒力因子与细菌群体感应(quorum-sensing, QS)相关基因表达量；利用分子对接技术检测大蒜素与群体感应关键蛋白的结合力。结果 相较于对照组，大蒜素处理组细菌的早期生长速率无明显差异，在生长中后期随大蒜素浓度的增加，生长速率逐渐降低；大蒜素对肺炎克雷伯氏菌的MIC和MBC分别为200 μg/mL和800 μg/mL。显微镜观察发现，50 μg/mL大蒜素处理导致膜结构损伤，使细菌形态由短杆状转变为球状；并且细菌的膜电势和膜通透性随大蒜素处理浓度的升高而逐渐增加。此外，大蒜素各处理组中细菌荚膜的形成量均显著降低；两组的生物膜形成量受到明显抑制，25 μg/mL与50 μg/mL组无明显差异；并且50 μg/mL处理组的Ⅰ型菌毛合成明显下调。RT-qPCR结果表明，荚膜(rmpA, magA)、Ⅰ型菌毛(fimD, fimK)、生物膜(RcsB, bsmA)合成相关基因均下调表达；群体感应相关基因(LuxS, LsrK)的表达量也降低，并且分子对接显示大蒜素可与LuxS蛋白和LsrK蛋白结合互作。结论 大蒜素通过细菌群体感应途径影响肺炎克雷伯氏菌的生长与生物膜、荚膜、Ⅰ型菌毛等毒力表型。本研究为大蒜素作为潜在药物抑制肺炎克雷伯氏菌应用及作用机制提供了理论依据。

Abstract:

Background Klebsiella pneumoniae is a common pathogen, whereas drugs are limited for its clinical prevention and treatment. Screening and developing new drugs is particularly important for the prevention and treatment of this pathogen. Allicin with multiple pharmacological functions has been studied regarding its antibacterial effects. The effect of allicin on the virulence of K. pneumoniae, however, has been rarely reported.Objective To study the effects of allicin on the growth and virulence factors of K. pneumoniae and decipher the underlying mechanism.Methods We inoculated K. pneumoniae in the liquid media containing different concentrations (25 μg/mL and 50 μg/mL) of allicin to plot the growth curve and determine the survival rate. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of allicin were determined by the microbroth dilution method. The morphology of bacteria was observed in the microscope, and the membrane potential and permeability were measured. The formation of biofilm, capsules, and fimbriae was detected. Furthermore, RT-qPCR was employed to determine the expression of virulence genes and quorum sensing (QS)-related genes. The binding of allicin to key proteins of QS was examined by molecular docking.Results Compared with the control group, the two allicin treatment groups showed no significant changes in the early growth rate of bacteria. The bacterial growth rate in the middle and late stages gradually decreased as the allicin concentration increased. The MIC and MBC of allicin against K. pneumoniae were 200 μg/mL and 800 μg/mL, respectively. Microscopic observation revealed that 50 μg/mL allicin resulted in damage to the membrane structure, changing the bacteria from short rods to spheres. The membrane potential and permeability of K. pneumoniae enhanced gradually with the increase in allicin concentration. Moreover, the capsule formation of K. pneumoniae was markedly diminished in the two allicin treatment groups. Allicin significantly inhibited the biofilm formation of K. pneumoniae, with no significant difference between the 25 μg/mL and 50 μg/mL groups. The formation of type Ⅰ fimbriae in the 50 μg/mL allicin group was significantly inhibited. The RT-qPCR results indicated that the genes associated with the formation of capsules (rmpA and magA), type Ⅰ fimbriae (fimD and fimK), biofilms (RcsB and bsmA) exhibited down-regulated expression. The expression of QS-related genes LuxS and LsrK was also down-regulated. Molecular docking results showed that allicin bound to and interacted with LuxS and LsrK.Conclusion Allicin affected the growth and virulence phenotypes including biofilms, capsules, and type Ⅰ fimbriae of K. pneumoniae by regulating QS. This study provides a theoretical basis for elucidating the inhibitory effect and mechanism of allicin against K. pneumoniae.