Assembly of a model glycan uptake system from a symbiotic human gut bacterium

从共生人类肠道细菌中组装模型聚糖摄取系统

基本信息

批准号：
9078056
负责人：
Nicole M Koropatkin
金额：
$ 33.11万
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：
2016
资助国家：
美国
起止时间：
2016-06-06 至 2021-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9078056
关键词：
Affect Affinity Amino Acids Bacteria Bacteroides Bacteroides thetaiotaomicron Bacteroidetes Binding Binding Proteins Binding Sites Carbohydrates Cells Co-Immunoprecipitations Colitis Colorectal Cancer Complement Complex Data Development Diabetes Mellitus Digestion Disease Disease Outcome Ecosystem Escherichia coli Event Genome Goals Growth Health Homologous Gene Homologous Protein Human Image Immune Intestines Knowledge Life Lipoprotein Binding Maps Metabolism Modeling Molecular Molecular Structure Mutagenesis Nature Oligosaccharides Organism Pathway interactions Pattern Polysaccharides Process Property Proteins Proteomics Recombinants Site Starch Structure System Testing Work design extracellular fluorescence imaging gastrointestinal gut microbiota improved member microbial mutant nutrition pathogen protein structure public health relevance research study screening single molecule stoichiometry sugar targeted treatment unnatural amino acids uptake

项目摘要

DESCRIPTION (provided by applicant): The bacteria that inhabit the human intestinal tract are essential for immune and gastrointestinal development, pathogen protection, and complex carbohydrate digestion. Their ability to thrive in this niche is dependent upon their ability to extract carbohydrate nutrition from this highly competitive ecosystem. Bacteroidetes are numerically dominant Gram-negative members of the human gut microbiota that all rely upon similarly patterned outermembrane protein systems termed starch utilization (Sus)-like systems to capture carbohydrate nutrition. Every Sus-like system targets a unique glycan, and some species devote nearly 20% of their genomes towards encoding these proteins. Sus-like systems are only found in the Bacteroidetes, making these proteins attractive targets for manipulating the metabolism of these organisms to support human health. Our long-term goal is to understand the molecular events that support glycan utilization via Sus-like systems. In this proposal we will focus on the molecular interactions among the outermembrane proteins SusCDEFG in the prototypical starch utilization system (Sus) of Bacteroides thetaiotaomicron (Bt). We have determined the molecular structures of the starch-binding lipoproteins SusDEFG, but how these proteins interact with the TonB-dependent transporter SusC to facilitate glycan import is unknown. All Sus-like systems have homologs of SusC, and of SusD, and so understanding how SusCD interact with each other and with SusEFG will inform a general model of how Sus-like systems facilitate glycan uptake. In Specific Aim 1 we will determine the nature and stoichiometry of the SusCD interaction, as well as how SusEFG affect this assembly. Our working hypothesis is that SusD facilitates interactions between SusC and the SusEFG proteins. We will identify interacting Sus proteins via co-immunoprecipitation and proteomics. In parallel, we will examine the co- localization and stoichiometry of the Sus proteins in live cells y single molecule imaging. In Specific Aim 2 we will create a functional map of the SusC structure. Our working hypothesis is that the extracellular loops of SusC bind SusD. Although recombinant SusD has weak affinity for starch, the SusCD interaction may create a higher affinity site for maltooligosaccharides that enhances import. We will test targeted mutants of susC and susD in Bt and E. coli for their ability to interact with each other and starch. We will incorporate unnatual amino acids into SusC to introduce fluorescent tags and photo-crosslinkable residues that can map the interaction between SusC and SusD. Finally, we will determine the x-ray crystal structure of SusC with or without SusD to understand its topology and function. Together, these data will reveal the molecular details of the Sus complex assembly, and allow us to generate a working model of a conserved glycan acquisition paradigm that is unique to the Bacteroidetes. With these details we can design new and selective strategies to manipulate microbial metabolism in the human gut.

描述（由适用提供）：遗传人肠道的细菌对于免疫和胃肠道发育，病原体保护和复杂的碳氢化物消化至关重要。他们在这个利基市场中成长的能力取决于他们从这个竞争激烈的生态系统中提取碳氢营养的能力。细菌植物是人类肠道菌群的数值主导革兰氏阴性成员，它们都依赖于类似的构成图案化的OUTEREMBRANE蛋白系统，称为淀粉利用率（SUS）类似于捕获碳水化合物营养的系统。每个类似SUS的系统都瞄准了一个独特的聚糖，一些物种将其几乎20％的基因组投入编码这些蛋白质。 SUS样系统仅在细菌群中发现，使这些蛋白质具有吸引力的靶标，以操纵这些生物的代谢以支持人类健康。我们的长期目标是了解通过类似SUS的系统支持聚糖利用的分子事件。在这个建议中，我们将侧重于细菌型Thetaiotaomicron（BT）的原型淀粉利用系统（SUS）中外膜蛋白之间的分子相互作用。我们已经确定了星形结合脂蛋白susdefg的分子结构，但是这些蛋白如何与TONB依赖性转运蛋白相互作用以促进聚糖进口是未知的。所有类似SUS的系统都有SUSC和SUSD的同源物，因此了解SUSCD如何相互作用以及与SuseFG相互作用，将为SUS样系统如何促进聚糖的吸收方式提供通用模型。在特定目标1中，我们将确定SUSCD相互作用的性质和化学计量，以及SuseFG如何影响该组件。我们的工作假设是SUSD的最爱相互作用与SUSEFG蛋白之间的相互作用。我们将通过共免疫沉淀和蛋白质组学鉴定相互作用的SUS蛋白质。同时，我们将检查活细胞y单分子成像中SUS蛋白的合作定位和化学计量。在特定目标2中，我们将创建SUSC结构的功能图。我们的工作假设是SUSC的细胞外回路结合了susd。尽管重组SUSD对淀粉的亲和力较弱，但SUSCD相互作用可能会产生更高的麦芽糖糖类亲和力位点，从而增强进口。我们将测试SUSC的靶向突变体，并在BT和E. Coli中的SUSD相互相互作用和淀粉的能力。我们将将不自然的氨基酸纳入SUSC中，以引入荧光标签和可将照片连接保留的保留，这些保留可以绘制SUSC和SUSD之间的相互作用。最后，我们将确定有或没有SUSC的SUSC的X射线晶体结构，以了解其拓扑和功能。这些数据将共同揭示SUS复合物组件的分子细节，并允许我们生成一个保守的Glycan采集范式的工作模型，该模型是细菌植物所特有的。有了这些细节，我们可以设计新的和选择性的策略来操纵人类肠道中的微生物代谢。