Sensing and Utilization of Mucopolysaccharides by Bacteroides thetaiotaomicron

描述（由申请人提供）：人类肠道微生物群提供了我们不必自行进化的生理属性，包括处理否则难以消化的膳食多糖的能力。拟杆菌（Bacteroides thetaiotaomicron， B. theta）是正常人类远端肠道微生物群的重要成员，具有处理膳食和宿主来源的多糖的能力，这一特征可能增强了其在拥挤的肠道生态系统中的适应性。一个基本的问题是这种原型肠道共生体如何识别和适应不断变化的碳水化合物供应。B. theta基因组编码50个胞质外功能sigma （ECF-?）因子，其中26个位于编码抗sigma （anti-?）因子的基因附近，这些基因是通过质周传感器结构域预测的跨膜蛋白。大多数ECF - ?/反?对（25/26）与第三个基因共定位，编码一个类似susc的外膜孔蛋白。SusC类似物与多糖结合有关，预计会直接与抗-？因子，包括一系列细胞包膜跨越开关，这些开关转导外部信号（SusC和anti-?）来影响转录变化（ECF-?）。我的体内和体外实验结果表明，B. theta通过ECF-?调节其生理机能来利用宿主来源的粘多糖。/反?相关的机制。我已经确定了四个原型基因座（44个基因），每个基因都含有一个ECF- /anti-？开关和多糖的分解代谢功能。我建议通过研究这4个系统的机制和信号转导的特异性以及它们在小鼠肠道中对B. theta适应度的贡献来探索这4个系统的功能。目标1将测试ECF-?/anti-?的工作模型。信号转导通过其预测成分的遗传破坏和随后的功能测定。酵母2杂交分析将用于探测信号组分之间预测的蛋白质-蛋白质相互作用，并且由于这些信号组分在B. theta中扩展，因此可能存在类似物之间的串扰。目的2试图从化学上定义触发ECF- /anti-?的粘多糖成分。与基因座诱导的生物测定相结合，将为B. theta感知环境的化学语言提供有价值的见解。目的3解决了B. theta利用粘多糖提高体内适应性的假设。B. theta开启这些系统的能力将通过基因操作消除，并且在与野生型B. theta的竞争中评估突变体的定植行为。这些研究将扩大我们对肠道细菌如何改变代谢以配合肠道中碳水化合物资源的变化的理解。

DESCRIPTION (provided by applicant): The human gut microbiota provide physiologic attributes that we have not had to evolve on our own, including the ability to process otherwise indigestible dietary polysaccharides. Bacteroides thetaiotaomicron (B. theta), a prominent member of the normal human distal gut microbiota, has an expanded capacity to process both dietary and host-derived polysaccharides, a feature that likely enhances its fitness in the crowded gut ecosystem. A fundamental question is how this prototypic gut symbiont recognizes and adapts to changing carbohydrate availability. The B. theta genome encodes 50 extra-cytoplasmic function sigma (ECF-?) factors, 26 are located adjacent to genes encoding anti-sigma (anti-?) factors, which are predicted transmembrane proteins with periplasmic sensor domains. Most ECF-? /anti-? pairs (25/26) co-localize with a third gene, encoding a SusC-like outer membrane porin. SusC paralogs are implicated in polysaccharide binding and are predicted to interact directly with anti-? factors, comprising a series of cell envelope- spanning switches that transduce external cues (SusC and anti-?) to effect transcriptional changes (ECF-?). My results from in vivo and in vitro experiments indicate that B. theta adapts its physiology to utilize host-derived mucopolysaccharides via an ECF-? /anti-? dependent mechanism. I have identified four prototypic loci (44 genes), each containing an ECF-?/anti-? switch and polysaccharide catabolic functions. I propose to probe the function of these 4 systems by investigating their mechanism(s) and specificity of signal transduction as well as their contribution B. theta fitness in the mouse intestine. Aim 1 will test a working model of ECF-?/anti-? signal transduction through genetic disruption of its predicted components and subsequent assay of function. Yeast 2-hybrid analysis will be used to probe predicted protein-protein interactions between signaling components and, because these signaling components are expanded in B. theta, the potential for cross-talk between paralogs. Aim 2 seeks to chemically define mucopolysaccharide components that trigger ECF-?/anti-? switches and, in conjunction with a bioassay for locus induction, will provide valuable insight into the chemical language through which B. theta perceives its environment. Aim 3 addresses the hypothesis that mucopolysaccharide utilization by B. theta enhances in vivo fitness. The ability of B. theta to turn on these systems will be eliminated through genetic manipulations, and the colonization behavior of mutants evaluated in competition with wild-type B. theta in gnotobiotic mice. These studies will expand our understanding of how bacteria inhabiting the gut can shift their metabolism to coincide with changing availability of carbohydrate resources in the intestine.