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，包括加工原本难以消化的膳食多糖的能力。多形拟杆菌（B. Theta）是正常人远端肠道微生物群的重要成员，具有扩大的加工膳食和宿主来源的多糖的能力，这一特征可能增强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可用性。B。θ基因组编码50个胞质外功能σ（ECF-？）因子，26位于邻近的基因编码反西格玛（反？）因子，其是具有周质传感器结构域的预测跨膜蛋白。大多数ECF-？/anti-？对（25/26）与第三个基因共定位，编码SusC样外膜孔蛋白。SusC旁系同源物涉及多糖结合，并预测直接与抗？因子，包括一系列的细胞包膜跨越开关，抑制外部线索（susC和抗？）以影响转录变化（ECF-？）。我的体内和体外实验结果表明，B。theta适应其生理利用主机衍生的粘多糖通过ECF-？/anti-？依赖机制我已经确定了四个原型基因座（44个基因），每个基因座包含一个ECF-？/反？开关和多糖分解代谢功能。我建议通过研究这4个系统的信号转导机制和特异性以及它们对B的贡献来探索它们的功能。theta适应性的研究目标1将测试ECF-？/反？通过其预测组分的遗传破坏和随后的功能测定进行信号转导。酵母双杂交分析将用于探测预测的信号传导组分之间的蛋白质-蛋白质相互作用，因为这些信号传导组分在B中扩增。theta，旁系同源物之间的潜在串扰。目的2寻求化学定义触发ECF-？/反？开关，并结合基因座诱导的生物测定，将提供有价值的洞察化学语言，通过B。theta感知环境目的3阐明了B利用粘多糖的假设。θ增强体内适应性。B的能力。将通过遗传操作消除开启这些系统的基因，并在与野生型B的竞争中评估突变体的定殖行为。theta在gnotobiotic小鼠。这些研究将扩大我们对肠道细菌如何改变其代谢以适应肠道中碳水化合物资源变化的理解。

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.