Fecal Phage: Exposing Unknown in the Dark Matter of the Human Gut

描述（由申请人提供）：病毒是地球上最丰富、最多样化、最不为人所知的生物实体。人类体内至少含有数万亿种病毒，其中大部分是噬菌体（噬菌体）感染胃肠道中的细菌。因为病毒利用宿主细胞进行繁殖，所以它们可以影响宿主群体。肠道噬菌体拥有的基因对它们的细菌宿主和它们所居住的人类的生物功能都是不可或缺的。虽然它们可能影响消化、致病性和免疫功能，但目前对这一关键群落知之甚少。该研究项目的目标是开发新的高通量方法来快速揭示和表征这种生物“暗物质”的多样性。在对四对双胞胎及其母亲的粪便病毒群落的初步研究中，我们发现80%的病毒基因组与已知序列几乎没有相似性。病毒基因进化迅速，使得基于同源性的序列功能搜索变得困难。我们的研究重点将是利用现有技术来表征这个未知的社区，包括阐明100个未知病毒基因的表型和结构。在目标1中，病毒宏基因组的遗传和功能多样性将被表征。将对存在于多人体内或长期稳定的病毒基因进行检查，重点关注那些可能具有影响宿主代谢功能的病毒基因。在Aim 2中，富含病毒衣壳和结构蛋白的样品的蛋白质组学将用于将这些蛋白质序列与DNA序列联系起来，并使用人工神经网络在宏基因组中搜索相似的序列。目的3侧重于表征可能影响宿主的代谢基因的功能。选择的100个序列将在大肠杆菌中表达，利用代谢阵列和代谢物质谱分析其对表型的影响，并确定其三维晶体结构，以寻找结构同源性而不是序列同源性。预计代谢基因的存在与人类宿主健康特征（如肥胖）之间的联系将被评估。结合起来，这些方法将大大增加我们对这些未被探索的病毒群落性质的认识。对人类肠道中未表征的病毒基因功能的理解不仅将揭示我们正在直接研究的双胞胎和母亲群体，还将解决有关人类健康与肠道病毒之间已知相关性的问题。这项研究可能会提高我们对病毒、细菌、人体代谢和免疫之间相互作用的基本理解。有关自身免疫疾病、代谢紊乱、过敏和许多其他疾病进展的发现可能成为可能。

DESCRIPTION (provided by applicant): Viruses are the most abundant, most diverse, and least understood biological entities on Earth. Humans contain at least several trillion viruses, largely phage (bacteriophage) infecting the bacteria in the gastrointestinal tract. Because viruses use their host cells to reproduce, they can impact the host populations. Gut phage possess genes which are integral to the biological functions of both their bacterial hosts and the humans they inhabit. While they potentially affect digestion, pathogenicity and immune function, currently, very little is known about this critical community. The goal of this research project isto develop novel high- throughput methods to rapidly reveal and characterize the diversity of this biological "dark matter." In the preliminary study of fecal viral communities from four pairs of twins and their mothers, we showed that 80% of viral genomes have little similarity to known sequences. Viral genes evolve rapidly, making homology- based searches for sequence function difficult. The focus of our research will be to characterize this unknown community using existing techniques, including the elucidation of the phenotype and structure of 100 unknown viral genes. In Aim 1, the genetic and functional diversity of the viral metagenomes will be characterized. Viral genes which are present in multiple people or stable through time will be examined, with a focus on those which are likely to have metabolic functions affecting the host. In Aim 2 proteomics of samples enriched for viral capsid and structural proteins will be used to link those protein sequences with the DNA sequences, and artificial neural networks will be used to search for similar sequences in the metagenomes. Aim 3 focuses on characterizing the function of metabolic genes that may affect the host. The 100 selected sequences will be expressed in E. coli, their effect on phenotype will be assayed with metabolic arrays and mass spectrometry of metabolites, and their 3D crystal structures will be determined in order to search for structural homology rather than sequence homology. The link between the presence of a predicted metabolic gene and human host health traits, such as obesity, will be evaluated. Combined, these approaches will greatly increase our knowledge of the nature of these unexplored viral communities. An understanding of uncharacterized viral gene function in the human gut will not only shed light on the twin- and-mother communities we are studying directly, it will also address questions about the known correlations between human health and gut viruses. This research will likely improve our fundamental understanding of the interactions of viruses, bacteria, human metabolism and immunity. Discoveries related to the progression of auto-immune diseases, metabolic disorders, allergies and many other conditions may become possible.