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

基本信息

批准号：
10198921
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
项目类别：
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/10198921
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/antagonist interest loss of function member microbial microbial community microbiota mutant novel pathogen pathogenic microbe prevent structural biology synergism tool transcriptome sequencing whole genome

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/antagonist interest loss of function member microbial microbial community microbiota mutant novel pathogen pathogenic microbe prevent structural biology synergism tool transcriptome sequencing whole genome

展开

项目摘要

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.

概括人类结肠中微生物群落（微生物群）的组成和生理学已连接多种疾病。大多数这些相互作用的机械细节仍然不太需要。大多数这些互动的共享细节是四个调查人员组装以进行拟议项目的重点是了解肠道微生物与分泌宿主粘液附着的复杂碳液相互作用并代谢复杂的碳水合物。粘液是将肠道细菌与宿主组织分离的第一个障碍物，并且是复杂的混合物分泌的粘蛋白糖蛋白和其他分子。一些细菌已演变为粘液上的觅食营养来源。我们先前已经表明，这种粘液觅食活性在外源性时增加饮食纤维多糖不存在。使用完全测序的人肠道细菌的gnotobiotic模型，我们已经表明，在纤维缺乏症中，肠道菌群诉诸于降解营养的粘液，导致侵蚀其完整性。在野生型小鼠中，降低的粘液屏障会增加上皮通道和致命性结肠炎通过粘膜病原体，柠檬酸啮齿动物。更引人注目的是，当相同的合成微生物群是在缺乏白介素10的小鼠中组装，一种细胞因子的功能损失与人类有关小儿炎症性肠病（IBD），动物在没有病原体的情况下会发出致命感染，但只有低纤维饮食。因此，我们的工作揭示了粘液完整性之间的功能连接，饮食和肠道微生物在沉淀IBD中。粘蛋白糖蛋白的完整解构需要酶联盟：宠物酶水解蛋白质主链，硫酸酶和糖苷在离散糖苷键内识别硫化或未剥离的寡糖和单糖的水解酶上下文。我们的中心假设是，粘蛋白通过单个活动在一系列顺序步骤中降解在这种酶浓缩物中，存在基本的催化步骤，这可能由不同的贡献协同作用可降解粘液的物种。我们将通过首先定义顺序来检验该假设降解所需的细菌酶的作用，位置特异性和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联合治疗纯重组酶，我们已经在合成微生物群的成员中鉴定出来。在并行，我们将衡量遗传学中个体，离散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纵模型物种。研究小组是由肠道细菌生理学和分子生物学学科中的四个领导者组成，结构生物学以及酶学，粘蛋白生物学和甘油分析，都对粘液机制具有共同的兴趣退化和对人类疾病的后果。这些实验的成功完成将定义细菌粘蛋白降解的一系列精确的机械步骤，并可能导致疗法限制这些疗法 IBD等疾病的事件。