乳杆菌表层蛋白的结构、性质及其对菌株益生特性的调控机制

Lactobacilli have been generally confirmed as probiotic strains, which can effectively inhibit the pathogen adhesion, invasion and colonization. Many research reported these probiotic properties are attributed to their surface layer proteins (SLPs). But SLPs from different genetic strains exhibit different structures and properties. Meanwhile, the effect and the regulation mechanisms of SLPs for the probiotic properties of lactobacilli strains are not clearly understood. In this project, several lactobacilli strains carrying SLPs are supposed to be selected and identified from the abundant resource. The structures and physicochemical properties of SLPs will be investigated by proteomics methods. The effects of SLPs on lactobacilli co-aggregation, surface properties and survival ability in the gastrointestinal tract are intended to be studied. The contribution of different SLPs to lactobacilli strains adhering intestinal epithelial cells and inhibiting pathogenic bacteria will be assessed through establishing human intestinal epithelial cell model. In order to reveal the regulatory mechanism of different SLPs for intestinal health, cellular and molecular biology techniques will be used to study the binding behavior between SLPs and extracellular matrix proteins, the competition, exclusion, and displacement effects of SLPs on the adhesion of intestinal pathogens to intestinal epithelial cells, as well as the inhibition of intestinal bacteria-induced cell apoptosis. In this project, biological contribution of SLPs to the probiotic properties of lactobacilli strains will be revealed in a new and unique perspective. And the relationship among SLPs, strain similarity and regulation mechanisms will be explored, which can provide a theoretical basis for the application of SLPs in the field of health interventions.

乳酸菌是一类公认的益生菌，可以有效抑制病原菌的黏附、侵袭和定植。大量研究显示，这与其可能携带的表层蛋白（SLP）有关。但不同亲缘菌株的SLP，其结构性质的异同，及对菌株益生特性的影响及调控机制尚不明确。本项目拟从丰富的乳酸菌资源库中甄选若干携带SLP的乳杆菌，应用蛋白质组学进行解析和比对；考察SLP对乳杆菌与病原菌共聚集能力、菌体表面性质和消化道耐受性等生理生化特性的影响；建立人肠上皮细胞模型，评估不同SLP在乳杆菌黏附肠上皮细胞以及抑制病原菌黏附和侵袭中的贡献；在细胞和分子水平上，研究SLP与细胞外基质蛋白的结合，对肠道病原菌胞壁质的作用，对病原菌黏附肠上皮细胞的竞争、排斥和替换作用，以及抑制病原菌引发的细胞凋亡的信号通路。本项目将从一个崭新而独特的视角来探索SLP对乳酸菌益生特性的生物学贡献，发掘SLP与菌株亲缘性及益生调控机制间的规律，为乳酸菌在健康干预领域的应用提供理论依据。

乳酸菌是一类公认的益生菌，可以有效抑制病原菌的黏附、侵袭和定植。大量研究显示，这与其可能携带的表层蛋白（SLP）有关。但不同亲缘菌株的SLP，其结构性质的异同，对菌株益生特性的影响及调控机制尚不明确。本项目从丰富的乳酸菌资源库中筛选出携带SLP的乳杆菌，采用蛋白质组学、分子生物学、细胞生物学以及免疫组化技术对来源不同的SLP的性质、结构和功能及其亲缘关系进行解析，并揭示其对菌株益生特性的调控机制。本研究以确认携带SLP的L. acidophilus NCFM为参照菌株，共筛选出分属9个属、15株携带SLP的乳杆菌，其相对分子质量均在45-47kDa之间。各菌株SLP氨基酸组成相似，且均含有31%~49%的疏水性氨基酸；两株L. gasseri和两株L. acidophilus的SLP二级结构组成相似，说明乳杆菌SLP二级结构与其菌株亲缘性相关。SLP对乳杆菌的存活率、表面疏水性、自动聚集能力、消化道耐受力等表面特性均有积极的贡献，且不同亲缘菌株的SLP贡献有差异。SLP还可通过排斥、竞争和替换的方式显著抑制E. coli和S. typhimurium对HT-29细胞的黏附，其中以排斥作用最强，替换作用最弱。L. acidophilus的表层蛋白可水解E. coli和S. typhimurium细胞壁肽聚糖，使病原菌细胞壁出现破损，处于亚致死状态，从而发挥其抑菌作用。 L. casei fb05、L. rhamnosus fb06、L. acidophilus fb11和L. acidophilus NCFM的SLP可通过稳定胞内线粒体膜电位和Ca2+浓度水平，抑制Caspase-1、Caspase-9和Caspase-3的激活，缓解E. coli和S. typhimurium引起的HT-29细胞染色质凝聚，从而降低E. coli和S. typhimurium导致的HT-29细胞凋亡损伤。这说明乳杆菌SLP可通过水解病原菌细胞壁、调控线粒体介导的凋亡通路，从而减少肠道致病菌引起的上皮细胞的凋亡与损伤。本项目从一个崭新而独特的视角探索了SLP对乳酸菌益生特性的生物学贡献，发掘了SLP与菌株亲缘性及益生调控机制间的规律，研究结果有助于推进和丰富人们对益生菌的进一步认知，为其在健康干预领域的应用提供了理论支撑。