Regulatory RNAs and RNA-binding proteins in Bacteroides thetaiotaomicron

细菌利用非编码rna和rna结合蛋白来维持细胞代谢，使全球基因表达适应不断变化的环境条件，感知营养物质，并保护自己免受噬菌体的侵害。细菌RNA的研究已经对生物医学和生物技术做出了根本性的贡献。然而，我们对细菌RNA生物学的大量知识源于对少数好氧模式生物的研究，而许多医学相关的共生物种的RNA生物学，例如构成我们肠道微生物群的众多厌氧菌，在很大程度上是一个未开发的领域。保守分析表明，在系统发育上遥远的细菌之间，rna介导的过程只有有限的重叠，并且许多调控rna是家族、属甚至菌株特异性的。这意味着，从研究密集分布在我们肠道中的共生生物的RNA生物学中，我们可以学到很多东西。拟杆菌（Bacteroides thetaiotaomicron）是一种主要的人类肠道厌氧共生体，有助于宿主代谢和对入侵病原体的定植抗性。先前的研究将成功的生态位定殖主要归因于利用多种膳食聚糖的能力。然而，对潜在调控网络的研究很少，迄今为止仅限于基于蛋白质的转录控制机制。与之形成鲜明对比的是，对于B. thetaiotaomicron通过小调控rna （small regulatory RNAs, sRNAs）及其相应的蛋白伴侣等方式对基因表达的转录后调控却知之甚少。我们最近重新注释了B. thetaiotaomicron转录组，发现了约200个新的候选sRNA；其中一些可以同时进行实验验证。我建议鉴定那些与其体内生态位定植和与宿主细胞相互作用相关的拟杆菌类sRNAs。为此，B. thetaiotaommicron原型菌株VP 5482将用于在缺氧条件下定植人大肠原代3D模型。在共培养的特定时间点，RNA将被提取并应用于宿主-微生物转录组分析。为了进行深入的机制表征，将根据宿主定殖过程中相应缺失突变体的表达模式、保守性和表型来选择候选sRNA。对于这些优先排序的srna，目标转录本将通过计算和实验筛选相结合来识别。与此同时，可能与选定的sRNA相互作用的rna结合蛋白将通过拉下适配体标记的sRNA变体来鉴定。sRNAs与其靶转录物和蛋白质结合伙伴相互作用的分子决定因素将通过各种生化分析发现。总之，该项目将确定体内相关的rna介导的控制机制，这些机制被我们肠道微生物群的一个关键成员用来定殖其生态位。

Bacteria employ noncoding RNAs and RNA-binding proteins to maintain cellular metabolism, adapt global gene expression to changing environmental conditions, sense nutrients, and protect themselves against bacteriophages. Bacterial RNA research has already made fundamental contributions to biomedicine and biotechnology. However, our bulk knowledge on bacterial RNA biology stems from the study of a small number of aerobic model organisms, whereas RNA biology in many medically relevant commensal species, e.g. the numerous anaerobic bacteria constituting our gut microbiota, is a largely unexplored field. Conservational analyses suggest that there is only limited overlap in RNA-mediated processes between phylogenetically distant bacteria, and that many regulatory RNAs are family-, genus-, or even strain-specific. This implies that there is much to be learned from studying RNA biology in the commensals that densely populate our intestine.Bacteroides thetaiotaomicron is a major anaerobic human gut symbiont that contributes to host metabolism and colonization resistance against invading pathogens. Previous studies have attributed successful niche colonization largely to the ability to utilize a wide array of dietary glycans. However, investigations of the underlying regulatory networks are sparse and have so far been restricted to protein-based transcriptional control mechanisms. In stark contrast, little is known about B. thetaiotaomicron post-transcriptional control of gene expression, executed e.g. by means of small regulatory RNAs (sRNAs) and their corresponding protein partners. We have recently re-annotated the B. thetaiotaomicron transcriptome and discovered ~200 novel sRNA candidates; several of which could meanwhile be experimentally validated.I propose to identify those Bacteroides sRNAs that are relevant for colonization of its in-vivo niche and for the interaction with host cells. To this end, B. thetaiotaomicron prototype strain VP 5482 will be used to colonize a human primary 3D model of the large intestine under hypoxic conditions. At defined time points of the co-culture, RNA will be extracted and applied to host-microbe transcriptome profiling. For in-depth mechanistic characterization, sRNA candidates will be selected based on their expression patterns, conservation and phenotype of a corresponding deletion mutant during host colonization. For these prioritized sRNAs, target transcripts will be identified by a combination of computational and experimental screens. In parallel, RNA-binding proteins potentially interacting with the selected sRNAs will be identified by pull-down of aptamer-tagged sRNA variants. The molecular determinants for the sRNAs to interact with their target transcripts and protein binding partners will be uncovered using a variety of biochemical assays. Together, this project will identify in vivo-relevant RNA-mediated control mechanisms used by one of the key members of our gut microbiota to colonize its niche.