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

揭示肠道细菌粘液降解的酶促途径以治疗炎症

• 批准号: 10424458
• 负责人: Nicole M Koropatkin
• 金额: $ 55.4万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10424458
• 关键词: Animals; Anti-Inflammatory Agents; Antigens; Bacteria; Bacterial Physiology; Bacteroides; Bacteroides thetaiotaomicron; Biological Assay; Biology; Calorimetry; Carbohydrates; Cell surface; Childhood; Chronic Disease; Citrobacter rodentium; Colitis; Colon; Colorectal Cancer; Communities; Complex; Complex Mixtures; Crystallography; Data; Development; Diet; Dietary Fiber; Discipline; Disease; Ecosystem; Enzymatic Biochemistry; Enzymes; Epithelial; Event; Family; Fiber; Future; Gene Expression Profiling; Genetic Polymorphism; Glycobiology; Glycoproteins; Glycoside Hydrolases; Glycosides; Gnotobiotic; Goals; Goblet Cells; Growth; Health; Human; In Vitro; Individual; Infection; Inflammation; Inflammatory Bowel Diseases; Interleukin-10; Intestines; Knowledge; Lead; Link; Measures; Microbial Physiology; Modeling; Molecular; Molecular Biology; Molecular Weight; Monosaccharides; Mucins; Mucous Membrane; Mucous body substance; Mus; Mutation; Nutrient; Oligonucleotides; Pathway interactions; Peptide Hydrolases; Play; Polysaccharides; Predisposition; Process; Proteins; Recombinants; Research; Research Personnel; Resort; Role; Series; Signal Transduction; Source; Specificity; Structure; Substrate Specificity; Sulfatases; Sulfate; Surface; System; Testing; Therapeutic; Thick; Tissues; Titrations; Vertebral column; Wild Type Mouse; Work; base; combinatorial; commensal microbes; cytokine; design; enzyme activity; experience; experimental study; fitness; gastrointestinal; genetic manipulation; glycosylation; gut bacteria; gut microbes; gut microbiota; host-microbe interactions; human disease; improved; in vivo; in vivo Model; inhibitor; interest; loss of function; member; microbial; microbial community; microbiota; mutant; novel; pathogen; pathogenic microbe; prevent; structural biology; synergism; tool; transcriptome sequencing; whole genome

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.

总结 人类结肠中微生物群落（微生物群）的组成和生理学已经联系起来， 对很多疾病的影响。大多数这些相互作用的机制细节仍然非常需要。共享 四名研究人员聚集在一起进行拟议的项目，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并代谢，特别是附着在分泌的宿主粘液上的聚糖。 粘液是将肠道细菌与宿主组织分离的第一道屏障，是一种复杂的混合物， 分泌的粘蛋白糖蛋白和其它分子。一些细菌已经进化到以粘液为食， 营养的来源。我们以前已经表明，这种粘液觅食活动增加时，外源性 不存在膳食纤维多糖。使用完全测序的人类肠道细菌的gnotobiotic模型，我们 已经表明，在纤维缺乏期间，肠道微生物群诉诸于降解粘液以获得营养，导致 侵蚀其完整性。在野生型小鼠中，减少的粘液屏障增加了上皮细胞的进入和致命的结肠炎 由粘膜病原体啮齿类柠檬酸杆菌引起。更引人注目的是，当同样的合成微生物群 在白细胞介素10缺陷的小鼠中组装，白细胞介素10是一种细胞因子， 儿科炎症性肠病（IBD），动物在没有病原体的情况下发展致命的炎症， 但只能吃低纤维食物因此，我们的工作揭示了粘液完整性， 饮食和肠道微生物在沉淀IBD。粘蛋白糖蛋白的完全解构需要一个 酶的集合体：水解蛋白质骨架的肽酶和硫酸酯酶和糖苷酶 识别离散糖苷键内硫酸化或非硫酸化寡糖和单糖水解酶 contexts.我们的中心假设是，粘蛋白是在一系列连续的步骤，由个人活动降解 在该酶集合体中存在必需催化步骤，这可能由不同的 协同作用降解粘液的物种。我们将通过首先定义连续的 作用，位置特异性和降解所需的细菌酶的关键结构方面， 胃肠道粘蛋白。我们将使用各种形式的粘蛋白的顺序和组合治疗， 纯的重组酶，我们已经在我们的合成微生物群的成员中鉴定出。在 同时，我们将测量在遗传学范围内单个、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型物种。研究小组正在 由肠道细菌生理学和分子生物学、结构生物学 和酶学，粘蛋白生物学和糖分析，所有这些都对粘液的机制感兴趣 退化和对人类疾病的后果。成功完成这些实验将定义 细菌粘蛋白降解的一系列精确的机械步骤，并可能导致治疗，以限制这些 IBD等疾病中发生的事件。