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

 描述（由申请方提供）：栖息在人体肠道中的细菌对于免疫和胃肠道发育、病原体保护和复杂碳水化合物消化至关重要。它们在这个生态位中茁壮成长的能力取决于它们从这个竞争激烈的生态系统中提取碳水化合物营养的能力。拟杆菌是人类肠道微生物群中数量上占优势的革兰氏阴性成员，它们都依赖于类似模式的外膜蛋白系统（称为淀粉利用（Sus）样系统）来捕获碳水化合物营养。每一个类Sus系统都针对一个独特的聚糖，一些物种将近20%的基因组用于编码这些蛋白质。Sus-like系统仅在拟杆菌中发现，使这些蛋白质成为操纵这些生物体代谢以支持人类健康的有吸引力的靶标。我们的长期目标是了解支持聚糖利用的分子事件，通过Sus-like系统。在本提案中，我们将 本论文主要研究多形拟杆菌（Bacteroides thetaiotaomicron，Bt）淀粉利用系统（Starch utilization system，Sus）外膜蛋白SusCDEFG之间的分子相互作用。我们已经确定了淀粉结合脂蛋白SusDEFG的分子结构，但这些蛋白质如何与TonB依赖性转运蛋白SusC相互作用，以促进聚糖的进口是未知的。所有的Sus-like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的相互作用将有助于了解Sus-like系统如何促进聚糖摄取的一般模型。在具体目标1中，我们将确定SusCD相互作用的性质和化学计量，以及SusEFG如何影响这种组装。我们的工作假设是，SusD促进SusC和SusEFG蛋白之间的相互作用。我们将通过免疫共沉淀和蛋白质组学鉴定相互作用的Sus蛋白。同时，我们将通过单分子成像检查Sus蛋白在活细胞中的共定位和化学计量。在具体目标2中，我们将创建一个SusC结构的功能图。我们的工作假设是，细胞外环的SusC结合SusD。尽管重组SusD对淀粉的亲和力较弱，但SusCD相互作用可产生对麦芽寡糖的更高亲和力位点，从而增强输入。我们将在Bt和E中测试susC和susD的靶向突变体。大肠杆菌的能力，相互作用和淀粉。我们将把非天然氨基酸引入到SusC中，引入荧光标记和可光交联的残基，可以映射SusC和SusD之间的相互作用。最后，我们将确定有或没有SusD的SusC的X射线晶体结构，以了解其拓扑结构和功能。总之，这些数据将揭示Sus复合物组装的分子细节，并使我们能够生成拟杆菌属特有的保守聚糖获取范例的工作模型。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。

 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.