The role of polysaccharide surface capsules in Bacteroides glycan degradation

描述（由申请人提供）：居住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（聚糖）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其栖息地的许多来自饮食和宿主的聚糖。拟杆菌门（Bacteroidetes）是肠道细菌中两个数量上占优势的门之一，其成员具有一系列同源的外膜蛋白系统（sus -样系统），可以结合并酶降解聚糖。这些物种普遍在细胞表面产生多种荚膜多糖（CPS）。这些胶囊的作用仍然不明确，尽管一些研究指出逃避或操纵宿主免疫。我们已经证明，在丰富的人类肠道共生体拟杆菌（Bt）中，CPS的表达与一些Sus样系统的表达是协调的，这些系统参与降解宿主来源的粘液聚糖。此外，被迫完全依赖非生物小鼠肠道中宿主聚糖的Bt群体表达的CPS与喂食富含植物聚糖的小鼠中的Bt群体表达的CPS不同。这些观察结果导致了我们的中心假设，即Bt的表面CPS结构与它正在分解代谢的特定聚糖协调表达，因为每个胶囊的生化特性与特定的聚糖营养物质子集兼容。几种性质可能导致这种现象，包括外源聚糖与某些CPS结构的混溶性增加，或者从降解的聚糖中提取的糖再循环到新的胶囊中。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基因表达的改变降低了其在某些底物（如粘液o -聚糖）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更快。为了扩展这些发现，我们构建了一系列的八个突变体，每个突变体除了一个CPS位点外都有缺陷。每个菌株只产生一个表面胶囊，使我们能够在体外和体内分离其对不同聚糖生长的贡献。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，来测量单个胶囊对体外Bt聚糖代谢的影响。此外，这些菌株提供了一个独特的机会来分离每种CPS聚合物并探索其糖化学结构，我们将与普渡大学的Bradley Reuhs博士合作。最后，我们将这八种菌株的核苷酸标记变体引入无菌小鼠，以测量它们在体内相互竞争的能力。我们将操纵饲喂给小鼠的饮食聚糖的类型和丰度，并检查每个菌株黏液层的定植，作为我们假设将影响单个表达cps菌株的适应度的两个变量。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体肠道中组装成复杂且生理活跃的群落的日益加深的理解中。

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.