Regulatory RNAs and RNA-binding proteins in Bacteroides thetaiotaomicron

多形拟杆菌中的调节性 RNA 和 RNA 结合蛋白

• 批准号: 436382683
• 负责人: Professor Dr. Alexander Westermann, Ph.D.
• 金额: --
• 依托单位: Biozentrum der Universität Würzburg
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/436382683?language=en
• 关键词: Regulatory; RNAs; RNA; binding; proteins

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.

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研究已经为生物医学和生物技术做出了重要贡献。然而，我们对细菌RNA生物学的大量知识源于对少量需氧模式生物的研究，而许多医学相关的微生物物种中的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探索的领域。保守性分析表明，系统发育上遥远的细菌之间的RNA介导过程仅存在有限的重叠，并且许多调节RNA是科、属甚至菌株特异性的。多形拟杆菌（Bacteroides thetaiotaomicron）是一种主要的厌氧人类肠道共生体，它有助于宿主代谢和抵抗入侵病原体的定植。先前的研究将成功的生态位定殖主要归因于利用广泛的膳食聚糖的能力。然而，调查的基础监管网络是稀疏的，到目前为止，仅限于蛋白质为基础的转录控制机制。与此形成鲜明对比的是，人们对B知之甚少。基因表达的多形核转录后控制，例如通过小调节RNA（sRNA）及其相应的蛋白质配偶体来执行。我们最近重新注释了B。thetaiotaomicron转录组，发现了约200个新的sRNA候选人，其中几个可以同时实验validated.I建议，以确定这些拟杆菌sRNA的定植相关的其体内生态位和与宿主细胞的相互作用。为此，B。多形微粒原型菌株VP 5482将用于在缺氧条件下定殖大肠的人原代3D模型。在共培养的规定时间点，提取RNA并应用于宿主-微生物转录组分析。对于深入的机制表征，将基于它们在宿主定殖期间相应缺失突变体的表达模式、保守性和表型来选择sRNA候选物。对于这些优先排序的sRNA，将通过计算和实验筛选的组合来鉴定靶转录物。同时，将通过拉下适体标记的sRNA变体来鉴定可能与所选sRNA相互作用的RNA结合蛋白。sRNA与其靶转录物和蛋白质结合伴侣相互作用的分子决定簇将使用各种生物化学测定法来揭示。总之，该项目将确定体内相关的RNA介导的控制机制，这些机制是我们肠道微生物群的关键成员之一用来殖民其生态位的。