Unraveling the enzymatic pathway of gut bacterial mucus degradation to treat inflammation

摘要 人类结肠中微生物群落(微生物区系)的组成和生理学已经联系在一起。 许多疾病的致病因素。这些互动中的大多数仍然迫切需要机械性的细节。共享的 聚集在一起进行这项拟议项目的四名研究人员的重点是了解肠道微生物如何 与复杂的碳水化合物相互作用和代谢--特别是附着在分泌的宿主粘液上的多糖。 粘液是将肠道细菌与宿主组织隔开的第一道屏障，是一种复杂的 分泌粘蛋白、糖蛋白等分子。一些细菌已经进化到以粘液为食 营养素的来源。我们之前已经证明，当外源物质增加时，这种粘液觅食活动会增加。 膳食纤维多糖缺乏。使用全测序的人类肠道细菌的诺生菌模型，我们 已经表明，在纤维缺乏期间，肠道微生物区系求助于降解粘液以获取营养，导致 对其完整性的侵蚀。在野生型小鼠中，粘液屏障的减少增加了上皮性通路和致死性结肠炎 由粘膜病原体轮状柠檬酸杆菌引起。更引人注目的是，当同样的合成微生物群 在缺乏白介素10的小鼠体内组装，白介素10是一种与人类功能丧失相关的细胞因子 儿科炎症性肠病(IBD)，动物在无病原体的情况下发生致命性炎症， 但只能吃低纤维饮食。因此，我们的工作揭示了粘液完整性、 饮食和肠道微生物在IBD发病中的作用。粘蛋白糖蛋白的完全解构需要一个 酶联合体：降解蛋白质骨架的多肽酶、硫酸酯酶和糖苷 识别离散糖苷键上的硫化或非硫化低聚糖和单糖的水解酶 上下文。我们的中心假设是粘蛋白是通过个体活动以一系列连续的步骤降解的 在该酶联合体和基本催化步骤中，可以由不同 协同工作以降解粘液的物种。我们将通过首先定义序列来检验这一假设 降解黄曲霉毒素所需细菌酶的作用、位置特异性和关键结构特征 胃肠道粘液。我们将使用顺序和组合治疗各种形式的粘蛋白 纯重组酶，我们已经在我们的合成微生物群的成员中鉴定出了这种酶。在……里面 同时，我们将衡量在基因上单独、离散的粘液降解步骤的需求- 使用体外和小鼠体内模型作为读数的可操作模式物种。研究小组是 由肠道细菌生理学、分子生物学、结构生物学等学科的四位领军人物组成 以及酶学、粘蛋白生物学和糖分析，都对粘液的机制有共同的兴趣 退化及其对人类疾病的后果。这些实验的成功完成将定义 一系列精确的细菌粘蛋白降解的机械步骤，并可能导致限制这些 像IBD这样的疾病中的事件。

Summary The composition and physiology of the microbial community (microbiota) in the human colon has been linked to a number of diseases. Mechanistic details for most of these interactions are still badly needed. The shared focus of the four investigators assembled to conduct the proposed project is to understand how gut microbes interact with and metabolize complex carbohydrates—especially the glycans attached to secreted host mucus. Mucus is the first barrier that separates intestinal bacteria from host tissue and is a complex mixture of secreted mucin glycoprotein and other molecules. Some bacteria have evolved to forage on mucus as a source of nutrients. We have previously shown that this mucus foraging activity increases when exogenous dietary fiber polysaccharides are absent. Using a gnotobiotic model of fully sequenced human gut bacteria, we have shown that during fiber deficiency the gut microbiota resorts to degrading mucus for nutrients, leading to erosion of its integrity. In wild-type mice, a reduced mucus barrier increases epithelial access and lethal colitis by the mucosal pathogen, Citrobacter rodentium. More strikingly, when this same synthetic microbiota is assembled in mice deficient in interleukin 10, a cytokine for which loss of function is associated with human pediatric inflammatory bowel disease (IBD), animals develop lethal inflammation in the absence of pathogen, but only on a low fiber diet. Our work has therefore revealed functional connections between mucus integrity, diet and gut microbes in precipitating IBD. The complete deconstruction of mucin glycoproteins requires a consortium of enzymes: peptidases to hydrolyze the protein backbone and sulfatases and glycoside hydrolases that recognize sulfated or unsulfated oligo- and monosaccharides within discrete glycosidic linkage contexts. Our central hypothesis is that mucin is degraded in a series of sequential steps by individual activities in this enzyme consortium and that essential catalytic steps exist, which may be contributed by different species that work synergistically to degrade mucus. We will test this hypothesis by first defining the sequential action, positional specificity and key structural facets of bacterial enzymes required for degradation of gastrointestinal mucins. We will use sequential and combinatorial treatments of various forms of mucin with pure recombinant enzymes, which we have already identified in the members of our synthetic microbiota. In parallel, we will measure the requirement for individual, discrete mucus-degrading steps within genetically- manipulable model species using in vitro and mouse in vivo models as readouts. The research team is composed of four leaders in the disciplines of gut bacterial physiology and molecular biology, structural biology and enzymology, mucin biology and glycoanalytics, all with a shared interest in the mechanisms of mucus degradation and the consequences for human disease. Successful completion of these experiments will define a precise series of mechanistic steps for bacterial mucin degradation and could lead to therapies to limit these events in diseases like IBD.