长双歧杆菌BBMN68胆盐响应表面蛋白的黏附机制研究

Bifidobacteria strains exhibit important beneficial effects on host health. Adhesion to human intestinal cells and persistence in the human gut are crucial for bifidobacteria to exert their health-promoting effects. Cell surface-associated proteins act as key factors involved in host-cell interactions, which are regarded to play important roles in adhesion of bifidobacteria strains to the host intestine. The expression of bacteria surface proteins can be affected by bile signal from host intestinal tract where bifidobacteria colonize. Notably, our previous study has shown that the adhesion ability of Bifidobacterium longum BBMN68 was enhanced in response to bile treatment. Meanwhile the transcription level of sortase involved in the anchoring of proteins to the cell surface was up-regulated under bile stress condition. These results suggested that cell surface proteins responded to bile signal are closely associated with the increased adhesion ability of BBMN68. Therefore, iTRAQ high-throughput sequencing is employed to analyze the surfacome of BBMN68 under bile stress condition. Adhesion factors are identified by constructing mutant strains and assessing their adhesion ability. Then, the functional domains and receptors of adhesion factors are determined to reveal the interaction mechanism between the bile-induced surface proteins and the host intestine epithelium. Furthermore, bacterial one-hybrid system is used to determine the transcription factors that regulate the expression of adhesion in BBMN68, which will contribute to clarify the regulation mechanism of adhesion medicated by bile-induced surface proteins in BBMN68. These results will provide a theoretical basis for revealing the molecular mechanisms underlying the adaptation of bifidobacteria to the human intestinal tract.

双歧杆菌具有重要的益生功能，黏附定植人体肠道是其发挥益生作用的前提。表面蛋白是介导双歧杆菌与宿主肠道互作的关键因子，在菌株黏附定植至宿主肠道的过程中发挥重要功能。而表面蛋白的表达受肠道环境中信号分子胆盐的影响，本课题组前期研究显示胆盐胁迫后长双歧杆菌BBMN68黏附能力提高，同时介导表面蛋白锚定的分选酶转录水平上调，表明胆盐响应的表面蛋白与BBMN68黏附能力提高紧密相关。因此，本课题首先利用iTRAQ高通量测序技术确定胆盐响应表面蛋白，通过对表面蛋白突变株黏附能力评价鉴定BBMN68关键黏附因子。在此基础上，对黏附因子功能结构域及其识别的宿主表面受体进行研究，明确双歧杆菌胆盐响应的黏附因子与肠道上皮细胞的互作机制；利用细菌单杂交系统筛选调控黏附因子表达的转录因子，阐明胆盐响应表面蛋白介导双歧杆菌黏附的调控机制。研究结果同时为从分子水平上揭示双歧杆菌肠道适应机制提供理论依据。

黏附定植人体肠道是双歧杆菌发挥益生作用的前提，表面蛋白是介导双歧杆菌与宿主肠道互作的关键因子。而表面蛋白的表达受肠道环境中信号分子胆盐的影响，本课题组前期研究显示胆盐胁迫后长双歧杆菌BBMN68黏附能力提高28倍，同时介导表面蛋白锚定的分选酶转录水平上调，表明胆盐响应的表面蛋白与BBMN68黏附能力提高紧密相关。因此，本课题首先利用TMT高通量测序技术筛选胆盐响应表面蛋白，共确定了菌毛蛋白FimB、三角形四肽重复蛋白TPR、CAMP因子和5’-甲硫腺苷和延伸因子EF-G共5个表面黏附蛋白响应胆盐信号上调表达，进一步利用透射电镜实验证明了包含FimB的菌毛Pil1响应胆盐后，菌毛数量不仅增加，而且菌毛长度延长至3-4 μm。乳酸乳球菌异源表达Pil1后黏附能力提高了8.9倍，进一步利用抗体封闭胆盐处理的长双歧杆菌BBMN68后，菌体的黏附率下降了82%，证明Pil1是促进胆盐处理菌株黏附能力提高的主要因素。为进一步全面揭示长双歧杆菌BBMN68的黏附机制，本研究通过生物信息学预测了长双歧杆菌中21个表面黏附因子，利用乳酸乳球菌的异源表达验证预测的黏附因子的功能，我们发现了一个全新的双歧杆菌表面黏附因子FimM，黏附受体筛选实验结合抗体封闭实验证明了纤连蛋白、纤维蛋白原和黏液蛋白是FimM在肠道中的特异性黏附受体。免疫金电镜实验确定FimM以单体蛋白的形式在细胞表面发挥黏附功能。FimM的异源表达使宿主菌的自聚集能力提高了47.7%，证明其具有促进肠道定植的作用。本课题的研究结果为从分子水平上揭示双歧 杆菌肠道适应机制提供理论依据。