High-Resolution Mapping of Bacterial Transcriptional Responses in Human-Associated Microbiota

项目摘要 微生物群落的功能分析对于了解它们对宿主健康的总体影响至关重要。大多数时候， 微生物组样品的宏基因组鸟枪测序用于评估总功能能力。然而，转录 不同生物体的反应可能会因环境不同而有很大差异，并可能产生很大影响。许多 代谢功能只有在生物体敏锐地感觉到它们体内特定底物的存在后才能表达。 环境病原体可能只在获得临界数量的病原体后才表达毒力因子。总的来说，压力 在不断变化的非生物和生物条件下，反应对生存至关重要。能够全面地绘制出 这些决定微生物组的弹性、可塑性和病理功能的途径，需要敏感的， 强大的转录组学工具。 对细菌群落进行传统的RNAseq分析一直是获得生物多样性的主要方法。 转录信息，但它受到技术变通方法的需要的阻碍，它提供了不完整的 关于转录景观的信息。核糖体RNA需要在测序之前耗尽，其具有差的 信噪比由不同的RNA衰变率引起，并且它对非编码RNA的转录不敏感 具有二级结构或转录后修饰的基因。或者，RNA聚合酶（RNAP）的位置 可以被评估，这提供了转录的实时读数。虽然所谓的新生转录测序已经 在E. coli，揭示了单独使用RNAseq时难以理解的转录暂停位点和其他现象， 这些方案依赖于RNAP的免疫沉淀，因此不适合复杂的微生物群落 其中RNAP可能是相当多样的，并且需要物种特异性抗体。作为解决方案，Precision Run-On和 测序（PRO-seq），最初创建用于检查真核生物中转录的方法，可以提供无偏见的 一种检测培养细菌或复杂微生物群落中转录动力学的方法，例如 人类微生物组。 我们的目标是测试PRO-seq应用于原核生物的可行性，并评估其捕获 转录动力学与典型的应激反应途径（热休克，氧暴露和DNA 损害），使用一组定量度量。我们的目标是验证，如果必要的话，修改协议，使其可以使用 在不同物种间都很活跃。我们计划开发一种计算方法来测试转录现象的全部广度 这种方法可以观察到转录暂停、双向转录、RNAP差异等 在物种间的功能和RNA衰变率等方面。如果成功，我们预计PRO-seq将 用于研究人类相关微生物群对宿主饮食，炎症信号，外源性物质的反应， 更直接地说，是人类的转录电路。

Project Summary Functional profiling of microbial communities is critical to understanding their overall effects on host health. Most often, metagenomic shotgun sequencing of microbiome samples is used to assess total functional capacity. Yet, transcriptional responses may vary dramatically between organisms depending on the context, with potentially large effects. Many metabolic functions are only expressed after the organism acutely senses the presence of particular substrates in their environment. Pathogens may only express virulence factors after obtaining a critical quorum of pathogens. Overall, stress responses are critical for survival under changing abiotic and biotic conditions. Being able to comprehensively map out these pathways, which determine the resilience, plasticity, and patho-functions of the microbiome, requires sensitive, robust transcriptional –omics tools. Performing traditional RNAseq analyses on bacterial communities has been the predominant method to gain transcriptional information, but it is hampered by the need for technical workarounds and it provides incomplete information about the transcriptional landscape. Ribosomal RNA needs to be depleted prior to sequencing, it has a poor signal-to-noise ratio arising from varying RNA decay rates, and it is insensitive to the transcription of non-coding RNA that has secondary structure or post-transcriptional modifications. Alternatively, the position of RNA polymerase (RNAP) can be assessed, which provides a real-time readout of transcription. Although so-called nascent transcript sequencing has been performed in E. coli, revealing transcriptional pause sites and other phenomenon elusive when using RNAseq alone, these protocols rely on immunoprecipitation of RNAP and are therefore unsuitable for complex microbial communities where RNAP may be quite diverse and require species-specific antibodies. As a solution, Precision Run-On and SEQuencing (PRO-seq), a method originally created for examining transcription in eukaryotes, may provide an unbiased method to examine transcriptional dynamics on cultured bacteria or in complex microbial communities, such as the human microbiome. Our goal is to test the feasibility of PRO-seq when applied to prokaryotes and to evaluate its ability to capture transcriptional dynamics associated with canonical stress response pathways (heat-shock, oxygen exposure and DNA damage), using a set of quantitative metrics. We aim to validate, and if necessary, modify the protocol so it can be used robustly across species. We plan to develop a computational approach to test the full breadth of transcriptional phenomena that can be observed using this method, such as transcriptional pausing, bidirectional transcription, differences in RNAP function apparent across species, and RNA decay rates, among other aspects. If successful, we expect that PRO-seq will be adopted to study the responses of human-associated microbiota to host diet, inflammatory signals, xenobiotics and to human transcriptional circuitry, more directly.