乳酸杆菌持家蛋白GAPDH兼职肠道粘附和锚定功能的机制

Adhesive ability is basis for lactobacilli to colonize in intestine and benefit for healthy improvement. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is cytoplasmic enzyme which considered to be a housekeeping molecule. This enzyme re-associates with the cell walls after secretion and functions as a moonlighting protein involved in adhesion to gastrointestinal mucin and anchoring plasminogen, which plays a key role in colonization of lactobacilli in intestinal tract. However, the mechanism about GAPDH secretion and interaction with mucin and plasminogen has remained unknown. In this study, quantity and location of GAPDH on surface of lactobacilli was evaluated by immune electron microscopy with the corresponding antibody, membrane permeabilization after labeling with propidium iodide (PI) or diacetoxyfluoran (FDA) was evaluated by flow cytometry, regular pattern of secretion of GAPDH was concluded by relating the surface location and membrane integrity in growth stage of bacterium. A kinetics analysis was performed to determine the affinity between GAPDH and intestinal mucin of pig or oligosaccharide probe by quantitative surface plasmon resonace (SPR). With biotinylated or isotope 125I labeled plasminogen, the capacity of GAPDH was analyzed in binding and enhancing activation of plasminongen. The key site of amino acid was determined in adhesion to intestinal tract by evaluating the relationship between selected site-directed mutagenesis and adhesive ability of GAPDH. The aim is to find out what moonlighting function that the GAPDH exerts in adhesion and anchoring, and mechanism of colonization of Lactobacillus in intestinal tract. The study will provide theory base for breeding and utilizing probiotic such as lactobacilli, which will bring great interesting to promote gastrointestinal health of animal.

乳酸杆菌粘附特性为肠道定植和益生作用的基础。乳酸杆菌持家蛋白甘油醛-3-磷酸脱氢酶（GAPDH）由胞浆分泌到细胞表面，获得兼职蛋白功能——粘附肠道粘液素和锚定纤溶酶原并促进激活，在菌株肠道定植发挥关键作用。然而，GAPDH分泌规律、与肠道粘液素和纤溶酶原互作的分子机制仍然不明。本研究以免疫电镜分析GAPDH在乳酸杆菌表面含量、分布，流式细胞仪结合荧光标记检测细胞膜通透性，解析GAPDH跨膜分泌规律；采用表面等离子共振技术结合粘液素糖基探针，分析GAPDH对猪肠道粘附素粘附作用及其动力学特征；利用纤溶酶原生物素和同位素标记，分析GAPDH对纤溶酶原的锚定与促进转化激活作用；通过基因点位突变表达，分析GAPDH粘附功能的关键氨基酸位点。旨在揭示GAPDH兼职肠道粘附与锚定功能作用，阐明乳酸杆菌肠道定植的分子机制，为开发和利用高效乳酸杆菌等益生菌提供理论基础，对促进畜禽健康具有重要意义。

乳酸杆菌对肠道粘附是其在肠道内成功定植发挥益生性的基础。试验以具有高粘力乳酸杆菌ZJ617和低粘附性乳酸杆菌ZJ615为代表，分析ZJ615和ZJ617不同培养时间表面蛋白GAPDH含量、细胞膜通透性及两者关系；分析抗体封闭GAPDH、高碘酸氧化粘液素或双重处理，乳酸杆菌对粘液素粘附数量；Octet分子互作仪分析不同处理条件下乳酸杆菌与粘液素的动力学结合特征；比较蛋白组分析表明蛋白差异，仔猪动物试验分析乳酸杆菌对肠道粘附和益生效果。研究结果表明：1）乳酸杆菌表面蛋白GAPDH含量在6h（对数生长期）最高（P＜0.05），进入生长后期（12h、15h、18h）后含量显著下降（P＜0.05），高黏附乳酸杆菌ZJ617不同培养时间的GAPDH蛋白含量均高于ZJ615（P＜0.05）；乳酸杆菌培养至对数生长期（6h）细胞膜通透性最高（P＜0.05），生长后期（12h、15h、18h）通透性显示降低（P＜0.05）；高粘附乳酸杆菌ZJ617不同时间细胞膜通透性显著高于ZJ615（P＜0.05）；ZJ617和ZJ615不同培养时间表面蛋白GAPDH分泌量和细胞膜通透性相关性分别为0.8和0.6。2）抗体封闭GAPDH、高碘酸氧化粘液素，乳酸杆菌粘附数量显著降低（P＜0.05）；抗体封闭和高碘酸氧化双重处理，乳酸杆菌粘附数量进一步显著降低（P＜0.05）；上述各处理下，高粘附乳酸杆菌ZJ617粘附数量显著高于低黏附ZJ615（P＜0.05）。3）动力学相互作用结果显示，与对照组相比，高碘酸氧化、去蛋白、加热和抗体封闭理组之后，各组反应值减弱。4）乳酸杆菌表面蛋白的差异蛋白组学分析显示，与ZJ615相比，ZJ617有31个下调蛋白，90个上调蛋白，其中GAPDH表达上调一倍以上，与前述结果一致，显示其可能与肠道粘附相关。5）仔猪试验表明，ZJ617对空肠、回肠和结肠的粘附显著高于ZJ615，并改善了空肠和回肠的绒毛结构，显著减低了仔猪腹泻。由此可见，表面蛋白GAPDH与乳酸杆菌肠道粘附具有相关性，其在乳酸杆菌粘附肠道和发挥益生作用中发挥重要作用。研究有效阐释了乳酸杆菌肠道定植的分子机制，为开发和利用高效乳酸杆菌等益生菌提供理论基础，对促进畜禽健康具有重要意义。