AI-2/LuxS群体感应对仔猪肠道乳酸杆菌生物被膜调控作用及机制

Biofilm in the intestinal tract of piglets is an important part of mucosal barrier and is regulated by quorum sensing (QS) which is involved in the regulation of multicellular behavior through communication via small molecules. However, the mechnisms of QS to reglate the biofilm formation in the intestinal tract were still poorly elucidated. As the most studied and used probotics, the lactobacillus was identified to have the AI-2/Luxs quorum sensing system, but the role of AI-2/Luxs in modulating the biofilm formation is also waiting for clarify in the intestinal tract. Based on the previous study on the lactobacillus isolated from the intestinal tract of piglets, the types and structures of signal molecules from the lactobacillus will be analyzed by biosensors combining with HPLC-MS and NMR. Secondly, an AI-2-deficient mutant of lactobacillus will be constructed by the transposon delivery system. The difference in adhesion and biofilm between the AI-2-deficient mutant and wild strains will be investigated to obtain the direct evidences of QS involved in the formation of biofilm in the intestinal tract of piglets. The metagenomic and metabolomic consequences of luxS inactivation was revealed to elucidate the functionality of QS through affecting the mucosal biofilm and microbial community in the intestinal of piglet. Finally, the difference in protein expression between the AI-deficient mutant and the wild strain will be analyzed by proteomic techniques to reveal the mechanism to modulate the biofilm formation and metabolome caused by QS. This study is aimed to clarify the role and mechanisms of AI-2/LuxS quorum sensing system in modulating the formation of mocusal biofilm and metabolomic in the intestinal tract. The study will provide theoretical basis for the development of new targeted regulation techniques of bacterial community and biofilm base on the control of the bacteria communication underlying the QS systems. The study will contribute to the healthy of food animal and the innovation of the animal production.

仔猪肠道生物被膜为粘膜屏障主要构成之一，群体感应（QS）为细菌间通过化学信号分子进行信息交流机制而参与生物被膜形成调控，然而，有关肠道生物被膜QS调控机制仍然不明。乳酸杆菌作为主要益生菌拥有AI-2/LuxS群体感应系统，但其对肠道生物被膜调节效应与机制更待探究。因此，本研究采用报告菌株法结合高效液相色谱-质谱和核磁共振技术对乳酸杆菌QS信号分子进行检测及结构分析；构建luxS基因缺失株，分析AI-2信号与乳酸杆菌定植仔猪肠道和形成生物被膜的相关性；采用宏基因组和代谢组学分析luxS缺失对菌株调节肠道菌群和代谢功能的影响及机制；运用蛋白质组学分析野生株和缺失株差异表达蛋白及与菌株肠道功能作用的关联机制。旨在阐明AI-2/LuxS群体感应调控乳酸杆菌肠道生物被膜形成与代谢效应的作用及机制，将为通过控制QS靶向调控肠道菌群与生物被膜提供理论基础，对创新动物健康调控技术具有重要启示意义。

仔猪培育是发展养猪生产的基础，培育品质优良的仔猪是提高猪肉品质的重要前提条件。仔猪由于消化器官不发达、肠道微生物区系不稳定和消化机能不完善等生理特点，导致肠道疾病和腹泻等问题。动物肠道是一个具有生物多样性的微环境，乳酸杆菌可通过竞争性粘附而形成生物膜等增强肠道屏障功能。仔猪肠道生物被膜为粘膜屏障主要构成之一，群体感应（QS）为细菌间通过化学信号分子进行信息交流机制而参与生物被膜形成调控，然而，有关肠道生物被膜QS调控机制仍然不明。乳酸杆菌作为主要益生菌拥有AI-2/LuxS群体感应系统，但其对肠道生物被膜调节效应与机制更待探究。.本研究建立了群体感应AI-2信号分子定性、定量检测方法；分析了早期断奶与正常哺乳仔猪粪便肠道菌群与AI-2的关联性，解析了乳酸杆菌对仔猪免疫调控作用及信号机制；构建了乳酸杆菌luxS基因缺失株，解明了AI-2信号分子对乳酸杆菌生长和生物膜形成的影响；阐明了AI-2/LuxS群体感应在调控宿主免疫中的重要作用及机制。研究发现，luxS缺失株中生物膜形成，细胞外多糖分泌和粘附都减弱了，外源加入AI-2信号分子能在一定程度上回补这些缺陷；AI-2/LuxS系统可能会影响生物膜的形成进并调节宿主免疫力；仔猪早期断奶肠道菌群的组成发生了显著的变化，肠道中的菌属Anaerovibrio，Prevotella，Bilophila等与群体感应呈显著相关，进而对仔猪的肠道健康产生了影响。本研究从群体感应信号分子作用角度深层次解析生物膜与微生态调控机制，为靶向控制群体感应的动物肠道健康调控新技术的开发提供新思路，对促进仔猪健康具有重要科学意义和指导作用。