大蒜精油活性组分对铜绿假单胞菌QS猝灭效应和分子机理研究

The extensive uses of antimicrobials have been providing sustained advantageous selection pressure for resistant bacteria. Then the high resistant and multi-drug resistant bacteria occurred frequently. Therefore humans need to change the prevention strategies of harmful microorganisms. Quorum quenching (QQ) has been suggested as a potential strategy for the control of harmful microorganisms. Many species of bacteria use quorum sensing (QS) to communicate with other bacteria in their environment. This communication can lead to activation of pathogenic factors and biofilm formation in some bacteria. The existence of some QQ matters in the microbes can attenuate their QS, leading to blocking some necessary gene expressions and pathogenic phenotypes. QQ can shut down the virulence expression in pathogenic bacteria rather than restrict cell growth. QQ has shown potential to overcome drug toxicities, complicated super-infection and antibiotic resistance. Our previous work showed that the garlic oil have QQ activity on Pseudomonas aeruginosa. However, the active ingredients of QQ activity in garlic oil, the QQ effects and molecular mechanism are all still unclear. Based on our previous studies, the goal of the proposal is to investigate the QQ effects and mechanism of active ingredients in garlic oil in cellular and molecular level with Pseudomonas aeruginosa PAO1 as the test strain. The study includes: ① Firstly, the active ingredients of QQ in garlic oil will be tracked with the QS reporting system. Then the QQ effects of active ingredients in garlic oil will be studied from three aspects including the bacterial structure, the secondary metabolites and the biofilm formation. Moreover, the dose–time effect and dynamics characteristics of the QQ effect of the active ingredients in garlic oil will be revealed. ② The key genes and proteins in all levels of the QS system composed of las, phl and PQS will be used as the main objects to study the QQ mechanism of the active ingredients in garlic oil on the level of transcription and final products. In this research, we aim to reveal the basic science problem of the QQ mechanism of garlic oil on bacteria. The project will be helpful to increase people's understanding on the regulatory process to the QS by the garlic oil, and will provide theory base for the research and development of garlic oil as QQ medicines and its application.

细菌群体感应（Quorum sensing，简称QS）的猝灭是一种重要的有害微生物治理策略。前期研究发现大蒜精油具有QS猝灭活性，但对其QS猝灭的活性成分、猝灭的效应和分子机理还基本是未知的。本项目在前期基础上，拟以铜绿假单胞菌PAO1菌株为受试菌，从细胞水平和分子水平研究大蒜精油活性组分的QS猝灭效应和机理，具体内容包括：①通过QS报告系统追踪大蒜精油QS猝灭的活性组分，并从菌体结构、次级代谢产物和生物膜形成的抑制效果几个方面分析大蒜精油活性组分的QS猝灭效应，揭示其剂量-时间效应和动力学变化特征；②针对QS系统中las、rhl和PQS各层级系统的关键基因和蛋白靶标，从转录水平和终产物水平研究大蒜精油活性组分对受试菌株QS猝灭的分子机制。本项目研究将揭示大蒜精油活性组分是如何通过抑制细菌群体感应的通路达到调控细菌毒力的重要科学问题，为大蒜精油作为细菌QS猝灭药物的研发和应用奠定理论基础。

抗生素的持续使用为高耐药菌株提供了持续有利的选择压力，加快了超级耐药和多重耐药菌株的形成，人类需要改变有害微生物的治理策略，寻找不同于抗生素或抗菌剂治理方法的新的作用靶标。近年来，细菌群体感应（Quorum sensing，简称QS）系统成了有害微生物治理的新的作用靶标。本项目以铜绿假单胞菌（Pseudomonas aeruginosa）PAO1为受试菌株，通过研究大蒜精油中活性化合物对该菌的QS抑制活性，发现大蒜精油中三种活性化合物二烯丙基二硫醚（diallyl disulfide，简称DADS）、烯丙基硫醚（diallyl sulfide，简称DAS）、二烯丙基三硫醚（diallyl trisulfide，简称DATS）均具有不同程度的QS抑制活性。项目采用生理生化实验技术、q-PCR技术、RNA高通量测序和iTRAQ蛋白质组测序分析技术相结合的研究方法，研究了大蒜精油中DADS对该菌QS系统的抑制机制。研究结果表明，DADS处理后，铜绿假单胞菌PAO1菌株QS的三个层级系统（las，rhl和pqs）中的所有关键基因（lasI、lasR、rhlI、rhlR、pqsA、pqsR）均表达下调，与之调控相关的毒力因子（包括弹性蛋白酶、绿脓菌素、细胞膜等）的相关基因也同时出现了表达下调。QS通路是该菌表达下调基因中最显著的通路。此外，DADS还抑制了该菌的细菌运动、向化性和双组分系统的关键基因的表达。这些实验结果表明，DADS通过抑制铜绿假单胞菌PAO1菌株的QS系统，进而抑制了该菌的毒力和致病力，同时降低了该菌对环境刺激的敏感性，使细胞处于一种不敏感的钝化状态。该项目的研究成果为铜绿假单胞菌的治理提供了新的途径，并为研发大蒜精油活性化合物的QS抑制剂奠定了理论基础。项目研究成果已经发表学术论文5篇，其中SCI收录论文5篇，并申请国家发明专利3件。