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

 描述(申请人提供)：居住在人体肠道的细菌对免疫和胃肠道发育、病原体保护和复杂的碳水化合物消化是必不可少的。它们在这一利基环境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区系中数量占主导地位的革兰氏阴性成员，它们都依赖于类似模式的外膜蛋白系统，称为淀粉利用(SUS)样系统来获取碳水化合物营养。每个SUS类系统都以一种独特的糖链为目标，一些物种将近20%的基因组用于编码这些蛋白质。SU类系统只在拟杆菌中发现，使这些蛋白质成为操纵这些生物的新陈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通过类SUS系统支持多糖利用的分子事件。在本提案中，我们将 重点研究了苏云金杆菌原型淀粉利用系统(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，但这些蛋白如何与TonB依赖的转运体SusC相互作用以促进多糖输入尚不清楚。所有的类SUS系统都有SusC和SusD的同源物，所以了解SusCD如何相互作用以及如何与SusEFG相互作用将为SUS类系统如何促进多糖摄取提供一个通用模型。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学，以及SusEFG如何影响该组装。我们的工作假设是，SusD促进了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来研究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SusC结构的功能图。我们的工作假设是SusC的胞外环与SusD结合。虽然重组的SusD对淀粉的亲和力很弱，但SusCD的相互作用可能会为麦芽低聚糖创造一个更高的亲和力位点，从而增强进口。我们将在Bt和E.Coli中测试susC和susD的靶向突变体，以了解它们相互作用和淀粉的能力。我们将在SusC中引入非天然氨基酸，以引入荧光标记和光交联残基，从而定位SusC和SusD之间的相互作用。最后，我们将测定有或没有有无悬浮物的晶体结构，以了解它的拓扑结构和功能。综上所述，这些数据将揭示SUS复合体组装的分子细节，并使我们能够产生一个保守的多糖获取范例的工作模型，这是类杆菌所特有的。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体肠道中的微生物新陈代谢。

 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.