Confining Single Cells to Enhance and Target Cultivation of Human Microbiome

描述(由申请人提供)：了解人类微生物群对于维持人类健康和预防疾病至关重要，但由于大多数微生物不能用传统方法培养，目前尚不清楚特定的微生物如何影响健康和疾病。需要既能提高培养微生物的成功率，又能针对具有高度生物医学兴趣的微生物的培养努力的技术。该项目将利用微流控技术，通过发展单细胞限流技术，克服传统培养和靶向方法的局限性。将单个细胞随机限制在小体积的液滴中(从皮升到纳升)将隔离微生物物种，并有可能通过启动单个细胞的高密度生长来培养新的微生物。这种单细胞限制技术产生的液滴可以分裂，对克隆姐妹种群进行并行的多个分析，从而能够进行杀死分析以识别一个姐妹种群中的微生物，并利用另一个姐妹种群进行生长。在这项技术中，为了针对生物医学感兴趣的微生物进行培养，将通过两种互补的方法识别新物种：基于基因的分析和基于功能的分析。根据现有的元基因组数据，基于基因的分析将识别所需的功能基因和16S序列，而基于功能的分析将识别所需的功能，即使它们与已知的基因序列无关。然后，确定的微生物物种将被用于扩大微菌落的规模，以使它们可用于测序和进一步研究。我们将开发和验证单细胞限制技术，使用来自人类结肠的硫还原菌作为测试系统。硫磺还原细菌具有很高的生物医学重要性，与溃疡性结肠炎和腹内感染有关，但仍知之甚少。我们将首先使用肠道衍生微生物的模型联盟，其中包含具有代表性的硫磺还原细菌Bilophila wadsworth ia，以开发和优化这项技术。接下来，我们将开发基于基因和基于功能的分析方法，并通过识别模型混合物中的硫酸盐还原细菌来测试它们。最后，我们将使用这些培养方法和检测方法从人的结肠中培养和筛选新的硫磺还原菌。这项技术将普遍适用于鉴定和培养人体肠道微生物群中的所有类别的微生物。该项目将通过开发和验证技术来影响生物医学科学和公共卫生，以增加我们对人类肠道微生物组中基因和功能之间的关系的了解，从而提高微生物对健康和疾病的贡献。 公共卫生相关性：声明微生物对胃肠道的功能至关重要。要了解它们在人类健康和疾病中的作用需要培养，但人类肠道微生物群中的大多数物种都很难培养。这项应用将开发基于受限的技术，以加强从人类结肠中培养微生物，并通过使用互补分析来识别具有高度生物医学兴趣的基因和功能的微生物，从而针对培养努力。

DESCRIPTION (provided by applicant): Understanding the human microbiome is critical in maintaining human health and preventing disease, but it has been unclear how specific microbes affect health and disease because the majority of microbes cannot be cultivated using traditional methods. Technologies are needed that can both increase the success rate for cultivating microbes and target cultivation efforts towards microbes of high biomedical interest. This project will use microfluidic confinement to overcome the limitations of traditional cultivation and targeting methods by developing "single cell confinement technology". Stochastic confinement of single cells in droplets of small volumes (picoliters to nanoliters) will isolate microbial species and, potentially, enable cultivation of new microbes by initiating high-density growth from a single cell. The droplets created by this single cell confinement technology can be split to perform multiple assays in parallel on clonal sister populations, enabling killing assays to be performed to identify microbes in one sister population, and using of the other sister population for growth. In this technology, to target cultivation efforts towards microbes of biomedical interest, new species will be identified via two complementary approaches: gene-based assays and function-based assays. Gene-based assays, informed by existing metagenomic data, will identify desired functional genes and 16S sequences, and function-based assays will identify desired functions even if they are not associated with a known gene sequence. The identified microbial species will then be targeted for scale-up of microcolonies to make them available for sequencing and further study. We will develop and validate the single cell confinement technology by using sulfur-reducing bacteria from the human colon as the test system. Sulfur-reducing bacteria are of high biomedical importance, associated with Ulcerative Colitis and intra-abdominal infections, but are still poorly understood. We will first use a model consortium of gut-derived microorganisms, containing a representative sulfur-reducing bacterium Bilophila wadsworthia, to develop and optimize the technology. Next, we will develop gene-based and function- based assays and test them by identifying sulfate reducing bacteria in model mixtures. Finally, we will use these cultivation approaches and assays to cultivate and select new sulfur-reducing bacteria from the human colon. This technology will be generally applicable to identify and cultivate all classes of microbes in the human gut microbiome. This project will impact biomedical science and public health by developing and validating technologies for increasing our understanding of the relationship between genes and functions in the human gut microbiome, and therefore microbial contributions to both health and disease. PUBLIC HEALTH RELEVANCE: Statement Microbes are critical to the function of the gastrointestinal tract. Understanding their role in human health and disease requires cultivation, but the majority of the species in the human gut microbiome are difficult to cultivate. This application will develop confined-based technology to enhance cultivation of microbes from human colon and target the cultivation efforts by using complementary assays to identify microbes with genes and functions of high biomedical interest.