Multi-method investigation and characterization of the ocular microbiome

人体内寄生着数以万亿计的共生细菌、古生菌和真菌，它们的总基因含量是 据估计，它们是人类宿主的2000多倍，其中大部分居住在肠道 并侵占许多其他身体部位，特别是皮肤和嘴巴。2007年启动的人类微生物组计划 在推动多组学工具的开发以表征微生物群方面发挥了重要作用， 然而，绝大多数研究都集中在对大肠微生物群的研究上。此外， 这些研究只是简单地对这些群落进行了排序，几乎没有努力去培养和 从功能上描述它们或它们各自的细菌菌株。因此，培养和表征 在微生物组领域，研究得比肠道更少的部位的微生物群落是一个关键的、尚未得到满足的需求 研究。眼表最初被认为是无菌的，因为很难获得有活性的微生物。 以传统培养为基础的技术，更不用说眼泪中存在的强大的抗微生物防御能力了。近期 研究表明，眼睛表面并不是无菌的，而是含有低生物量的微生物群落，这 这表明这些微生物可能在眼睛健康中发挥作用。然而，因为大多数研究都描述了 到目前为止，眼睛微生物组只专注于16S rDNA和元基因组测序，存在 真正的眼部微生物群仍然知之甚少。针对这一U24 RFA，我们建议更好地定义 眼表面的微生物和微生物群落的广度，通过组合 靶向培养和元基因组测序方法。一旦单个微生物和群落 经过培养和编目，我们将从功能上评估这些微生物对 人眼生物学。我们已经组建了一支多学科的团队，在眼科方面具有互补的专业知识 生物学、免疫学、微生物生态学和生理学、元基因组学和微生物培养产生 强大的微生物菌株、基因组、亚基因组和眼部及眼部功能谱系资源 微生物群落。在生成这些资源时要追求的以下目标是：目标1： 眼表面微生物群的培养及特性研究。我们将使用针对性的组合 培养、全长(FL)16S rDNA测序和全基因组鸟枪式(WGS)元基因组学 描述与眼表相关的微生物群，包括结膜、泪液和眼周 皮肤。目标2：微生物的功能特征和微生物群落与眼睛的相互作用。 为了了解眼睛相关微生物如何影响眼睛健康和生理，我们将结合培养 用原代人类细胞培养试验从眼表中分离出单个微生物以监测功能 微生物对眼睛的影响。我们的最终目标是在本建议书期末拥有一套强大的 科学界现在可以开始使用的具有良好特性的眼表微生物 眼睛/微生物组相互作用的特征。

The human body is host to trillions of commensal bacteria, archaea, and fungi whose total gene content is estimated to be over 2000 times more than that of their human hosts, most of which reside in the intestinal tract and colonize many other body sites, notably skin and mouth. The Human Microbiome Project, launched in 2007 by the NIH, has been instrumental in driving the development of multi-omic tools to characterize microbiomes, however the vast majority of studies have focused on studying the microbiome of the large intestine. Moreover, these studies have served to simply sequence these communities, with little effort put forth to cultivate and functionally characterize them or their individual bacterial strains. Therefore, cultivation and characterization of microbial communities at sites less well studied than the gut is a key, unmet need in the field of microbiome research. The ocular surface was initially thought to be sterile due to difficulty in obtaining viable microbes using traditional culture-based techniques, not to mention the potent anti-microbial defenses present in tears. Recent work has indicated the ocular surface is not sterile but contains a low biomass microbial community, which suggests these microbes may play a role in ocular health. However, because most studies characterizing the eye microbiome have focused solely on 16S rDNA and metagenomic sequencing to date, the presence of a bona fide ocular microbiome is still poorly understood. In response to this U24 RFA, we propose to better define the breadth of microbes and microbial communities that populate the ocular surface through a combination of targeted cultivation and metagenomic sequencing approaches. Once individual microbes and communities have been cultivated and cataloged, we will functionally assess the impact of these microbes on various aspects of human eye biology. We have assembled a multidisciplinary team with complementary expertise in ocular biology, immunology, microbial ecology and physiology, metagenomics and microbial cultivation to generate robust resources of microbial strains, genomes, metagenomes, and functional repertoire for the eye and microbiome communities. The following goals to be pursued in generating these resources are: Goal 1: Cultivation and characterization of the ocular surface microbiome. We will use a combination of targeted cultivation, full length (FL) 16S rDNA sequencing, and whole genome shotgun (WGS) metagenomics to characterize the microbiomes associated with the ocular surface including the conjunctiva, tears, and periocular skin. Goal 2: Functional characterization of microbes and microbial community interactions with the eye. To understand how eye associated microbes can impact eye health and physiology, we will combine cultivation of individual microbes from the ocular surface with primary human cell culture assays to monitor the functional impact of microbes on the eye. Our ultimate goal by the end of this proposal period is to have a robust set of well-characterized ocular surface microbes with which the scientific community can now begin functional characterization of eye/microbiome interactions.