乳杆菌群体感应信号分子干扰铜绿假单胞菌生物膜形成的机制研究

How to cure the multi-drug resistant Pseudomonas aeruginosa (PA) infection is a challenge. Biofilm formation, which is regulated by bacterial quorum sensing (QS), is a principal cause of the antibiotic resistance of PA. It was reported that the QS autoinducers of probiotics inhibited the biofilm formation of PA. However, it is still unclear about the mechanism of how probiotics QS interfering with the biofilm formation of PA. The scientific question is whether this interference of PA biofilm is related to the QS signal molecules interaction between these two microorganisms. To answer this question, it needs a controllable and detachable co-culture system to observe this process dynamically. Based on the foundation of researches on the probiotics microencapsulation, we found that the three-dimension culture system improved the expression of probiotic QS, which can facilitate autoinducers secretion and in situ release. It is beneficial to construct the co-culture system of probiotics and pathogen with microencapsulation method to investigate the interference of QS with biofilm formation. Therefore, this project proposes to make Lactobacillus microcapsules with microfluidic devices to realize the co-culture of Lactobacillus and PA, which can be used to real-time monitor the effect of signal molecules on biofilm formation of PA. This co-culture system is potential to study the effect of interactions of QS signal molecules on pathogenic biofilm formation, which is expected to reveal a new mechanism of probiotic treatment in drug-resistant pathogenic infection. This project will provide a new strategy for clinical treatment of drug-resistant pathogenic infection.

治疗多重耐药铜绿假单胞菌（PA）感染是临床上亟需解决的难题。群体感应（QS）调控的生物膜形成是PA产生耐药性的主要原因，研究表明益生菌的QS信号分子会抑制PA生物膜的形成。然而，缺乏明确的理论阐明益生菌QS对PA生物膜形成的干扰机制。这种抑制作用与两种微生物信号分子的互扰是否具有关联性？构建可控、易分离的共培养体系对该过程进行动态观测有望解决这一难题。前期研究中我们发现益生菌微囊内的三维培养环境不但使益生菌QS高表达，而且使QS信号分子大量富集并原位释放，有利于研究益生菌QS信号分子对病原菌的QS系统和生物膜形成的影响机制。因此，本项目拟利用微流控技术制备乳杆菌微囊，构建乳杆菌与PA的共培养体系，通过原位动态释放乳杆菌QS信号分子的方式，实时监测信号分子对PA生物膜形成的影响规律，探索QS信号分子相互作用机制。本项目有望揭示益生菌治疗耐药菌感染新机制，为临床上治疗耐药菌感染提供新策略。

如何治疗多药耐药细菌感染是临床上亟需解决的科学难题，新型抗生素的开发及新型抗菌策略的提出是攻克该问题的两种重要策略。本项目针对这两种策略共完成以下两部分内容：（1）基于益生菌天然代谢产物开发针对致病微生物的抗菌剂（2）利用纳米材料的类酶催化活性，靶向铜绿假单胞菌的群体感应（QS）系统，通过干扰微生物的通讯系统降低其毒副作用。获得如下重要结果：首先，本项目发现鼠李糖乳杆菌的代谢产物可以显著抑制铜绿假单胞菌、金黄色葡萄球菌、多药耐药大肠杆菌等致病菌的生长，长期使用过程（30天连续使用）中不会产生显著的耐药性，耐药指数（resistance dependent index）小于1。并且该代谢产物的生物相容性良好，有望成为抗生素的替代品应用于胃肠道细菌感染。其次，本项目开发了靶向铜绿假单胞菌的QS信号分子（高丝氨酸内酯类化合物，AHL）的纳米水解酶——Zn-N-C核心介孔碳材料，利用该材料的类酶催化活性，特异性水解AHL分子的内酯环使其失活，从而抑制铜绿假单胞菌生物膜的形成。最后，在纳米类酶活性筛选的过程中，意外的发现可以模拟脱氢酶的纳米材料——SnSe二维纳米片，该材料可以催化细胞内三羧酸循环中重要的脱氢反应，催化乳酸生成丙酮酸、催化乙醇生成乙醛、催化苹果酸生成草酰乙酸等。该成果在国际TOP期刊Angew. Chem. 上在线发表。以上成果的发现，为临床上对抗耐药细菌感染提供全新思路，若加强后续研究实现技术转化，有望带来巨大的经济效益。