The role of polysaccharide surface capsules in Bacteroides glycan degradation

描述(申请人提供)：栖息在人类远端肠道的微生物群落增加了我们消化复杂碳水化合物(多糖)的能力。这个群落中的细菌已经进化出几种策略来代谢淹没它们栖息地的许多饮食和宿主衍生的多糖。拟杆菌是肠道细菌的两个数量占主导地位的门之一，它拥有一系列同源的外膜蛋白系统(SUS类系统)，可以结合和酶降解多糖。这些物种在细胞表面无处不在地产生多个胶囊多糖(CP)。这些胶囊的作用尚不清楚，尽管一些研究指出，这些胶囊逃避或操纵宿主免疫。我们已经证明，CPS在丰富的人类肠道共生杆菌thetaiotaomicron(Bt)中的表达与一些参与降解宿主来源的粘液多糖的Sus类系统的表达是协调的。此外，被迫完全依赖灵知生菌小鼠肠道中的宿主多糖的Bt种群相对于喂食富含植物多糖的小鼠的Bt种群表现出不同的CP。这些观察结果导致了我们的中心假设，即Bt将其表面CPS结构的表达与它正在分解的特定多糖协调起来，因为每个单独胶囊的生化特性与特定的葡聚糖营养子集是相容的。几种特性可能导致这种现象，包括外源多糖与某些CPS结构的可混性增加，或从降解的多糖中提取的糖回收到新胶囊中。BT型菌株(VPI-5482)编码8个不同的基因簇来产生CPS。我们的初步数据表明，该菌株CPS基因表达的变化降低了其在某些底物如粘液O-多糖上的生长速度，而使其在其他底物上的生长速度与野生型相同或快于野生型。为了扩大这些发现，我们构建了一系列8个突变体，每个突变体除了一个CPS基因座外，其他所有基因都是缺陷的。每个菌株只产生一个表面胶囊，使我们能够分离出它在体外和体内不同的糖链上对生长的贡献。我们将使用这8个菌株，结合包含47种不同碳水化合物的定制生长阵列，来测量单独胶囊对体外Bt多糖代谢的影响。此外，这些菌株提供了分离每个CPS聚合物并探索其糖化学结构的独特机会，我们将与普渡大学的Bradley Ruhs博士合作进行这项工作。最后，我们将把这八种菌株的核苷酸标记变体引入无菌小鼠，以衡量它们在体内相互竞争的能力。我们将操纵喂给小鼠的饮食多糖的类型和丰度，并检查每个菌株对粘液层的定植，作为我们假设将影响单个表达CPS的菌株的适合性的两个变量。总之，在拟议的实验中收集的数据将使我们能够将可变CPS表达的作用整合到对细菌如何在人类肠道中组装成复杂的生理活性群落的日益增长的理解中。

DESCRIPTION (provided by applicant): The microbial community that inhabits the human distal gut increases our ability to digest complex carbohydrates (glycans). Bacteria in this community have evolved several strategies to metabolize the many diet- and host-derived glycans that inundate their habitat. Members of the Bacteroidetes, one of two numerically dominant phyla of gut bacteria, possess a series of homologous outer membrane protein systems (Sus-like systems) that bind and enzymatically degrade glycans. These species ubiquitously produce multiple capsular polysaccharides (CPS) on their cell surfaces. The role of these capsules remains undefined, although some studies point to evasion or manipulation of host immunity. We have shown that CPS expression in the abundant human gut symbiont Bacteroides thetaiotaomicron (Bt) is coordinated with expression of some Sus- like systems involved in degrading host-derived mucus glycans. Moreover, Bt populations that are forced to rely exclusively on host glycans in the intestines of gnotobiotic mice express different CPS relative to Bt living in mice fed a diet rich in plant glycans. These observations lead to our central hypothesis that Bt coordinates expression of its surface CPS structures with the particular glycan that it is catabolizing because the biochemical properties of each individual capsule are compatible with a specific subset of glycan nutrients. Several properties could contribute to this phenomenon, including increased miscibility of exogenous glycans with some CPS structures or recycling of sugars derived from degraded glycans into new capsules. The Bt type strain (VPI-5482) encodes eight different gene clusters for producing CPS. Our preliminary data suggest that alterations in CPS gene expression by this strain decrease its growth rate on some substrates such as mucus O-glycans, while rendering it identical to or faster than wild-type on others. To extend these findings, we have constructed a series of eight mutants that are each deficient in all but one CPS locus. Each strain produces only a single surface capsule, allowing us to isolate its contribution to growth on different glycans in vitro and in vivo. We wil use these eight strains, in conjunction with a custom growth array containing 47 different carbohydrates, to measure the effect of individual capsules on Bt glycan metabolism in vitro. In addition, these strains provide a unique opportunity to isolate each CPS polymer and explore its glycochemical structure, which we will perform in collaboration Dr. Bradley Reuhs from Purdue University. Finally, we will introduce nucleotide signature-tagged variants of these eight strains into germfree mice to measure their ability to compete against each other in vivo. We will manipulate the type and abundance of dietary glycans fed to mice, and examine colonization of the mucus layer by each strain, as two variables that we hypothesize will influence the fitness of individual CPS-expressing strains. Together, the data gathered in the proposed experiments will allow us to integrate the role of variable CPS expression into a growing understanding of how bacteria assemble into a complex and physiologically active community in the human intestinal tract.