Sensing and Utilization of Mucopolysaccharides by Bacteroides thetaiotaomicron

描述(由申请人提供)：人类肠道微生物区系提供了我们不必自己进化的生理属性，包括处理否则无法消化的饮食多糖的能力。Thetaiotaomicron(B.theta)是正常的人类肠道微生物区系中的一个重要成员，它具有处理膳食和宿主来源的多糖的扩展能力，这一特征可能会增强它在拥挤的肠道生态系统中的适应性。一个基本的问题是，这种典型的肠道共生体如何识别和适应不断变化的碳水化合物供应。西氏杆菌基因组编码50个胞外功能西格玛(ECF-？)26个因子位于编码抗Sigma(抗？)因子，预测具有周质感受器结构域的跨膜蛋白。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，并被预测直接与抗？因子，由一系列细胞包膜开关组成，这些开关传递外部提示(SusC和ANTI？)以实现转录改变(ECF-？)。我的体内和体外实验结果表明，B.theta通过ECF-/抗-？来适应其生理，以利用宿主来源的粘多糖。依赖机制。我已经确定了四个原型基因座(44个基因)，每个基因都包含一个ECF-？/抗？开关和多糖的分解代谢功能。我建议通过研究这四个系统的信号转导机制(S)和特异性以及它们在小鼠肠道中对B.theta适合性的贡献来探讨它们的功能。目标1将测试ECF-？/反？的工作模型。通过其预测成分的遗传干扰和随后的功能分析进行的信号转导。酵母双杂交分析将被用来探索预测的信号组件之间的蛋白质-蛋白质相互作用，因为这些信号组件在B.theta中扩展，所以可能在Paralog之间产生串扰。目标2寻求从化学角度定义可触发ECF？/抗？的粘多糖成分。开关，并结合位点诱导的生物测定，将提供对B.theta感知其环境的化学语言的宝贵洞察。目的3解决假设，即粘多糖利用B.theta提高在体内的适应性。B.theta开启这些系统的能力将通过基因操作被消除，突变体的定植行为在诺生菌小鼠中与野生型B.theta竞争进行评估。这些研究将扩大我们对肠道细菌如何改变其新陈代谢以适应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.