Antibody-mediated isolation and investigation of CPR bacteria

CPR 细菌的抗体介导分离和研究

• 批准号: 447383558
• 负责人: Dr. Marie Schölmerich
• 金额: --
• 依托单位: Department of Earth and Planetary Science
• 依托单位国家: 德国
• 项目类别: WBP Fellowship
• 财政年份: 2020
• 资助国家: 德国
• 起止时间: 2019-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/447383558?language=en
• 关键词: Antibody; mediated; isolation; investigation; CPR

The Candidate Phyla Radiation (CPR) bacteria account for an estimated 26 percent of the total catalogued biodiversity on Earth. Yet, nearly all major lineages within the CPR are lacking isolated representatives. A method shall be developed within this project to specifically capture, co-isolate and cultivate CPR bacteria with their host bacteria in the laboratory to gain first insights into their physiology. CPR bacteria are generally characterized by a small cell size, small genomes and are predicted to be anaerobes. They often lack essential biosynthetic clusters, leading to the prediction that they may require a symbiosis with other bacteria, archaea and even eukaryotes. However, almost all findings stem from genome-based observations so far. To assess the physiology and biochemistry of this monophyletic but metabolically diverse group, it is essential to develop techniques to isolate CPR representatives and co-cultivate them together with their host under laboratory conditions. To achieve this, environmental samples will be collected from a site close to the laboratory, which shows a high abundance in CPR bacteria. The cells from the site will be treated with fluorescently labeled antibodies which were raised against MAG- and SAG-derived extracellular domains of membrane proteins in CPR bacteria. The labeled cells will be sorted via flow cytometry, analyzed for CPR positives and cultivated under different anaerobic conditions. Cultivation assays containing growing CPR representatives will then be subjected to physiological analyses. The co-cultures of a CPR bacterium and its symbiotic host will be analyzed using fluorescence and cryogenic transmission electron microscopy to resolve the morphology and interaction of the CPR bacterium and its host. In a collaborative part of the project, the supernatant of growing CPR bacterial cultures will undergo first metabolomic analyses. The pure co-cultures will finally be used for genome sequencing and metabolic models will be constructed. The genome sequences will be screened for new antibody targets to optimize the antibody-facilitated capture of CPR bacteria. In summary, the project workflow is structured into the following three parts:Part A: Sample collection, processing, cell labeling and sorting of CPR bacteriaPart B: Cultivation and physiological and biochemical analyses of CPR isolatesPart C: Single cell genome sequencing, metabolic reconstruction and development of new antibodies for CPR captureThe workflow will be repeated using the newly developed antibodies to capture, isolate and characterize more CPR bacteria and assess the physiology and biochemistry of more CPR isolates. The project will thus result in a new tool to make CPR bacteria accessible for laboratory cultivations and shed first light onto the physiology of this widespread and yet enigmatic group of bacteria.

候选门辐射（CPR）细菌估计占地球上总编目生物多样性的26%。然而，几乎所有的主要血统在CPR缺乏孤立的代表。本项目应开发一种方法，在实验室中专门捕获、共分离和培养CPR细菌及其宿主细菌，以首次了解其生理学。CPR细菌通常以小细胞尺寸、小基因组为特征，并且被预测为厌氧菌。它们通常缺乏必要的生物合成簇，导致预测它们可能需要与其他细菌，古生菌甚至真核生物共生。然而，到目前为止，几乎所有的发现都来自于基于基因组的观察。为了评估这一单系但代谢多样性群体的生理和生物化学，必须开发分离CPR代表并在实验室条件下与宿主共同培养的技术。为了实现这一目标，将从实验室附近的一个地点收集环境样本，该地点显示CPR细菌丰度很高。来自该部位的细胞将用荧光标记的抗体处理，所述抗体针对CPR细菌中膜蛋白的MAG和SAG衍生的细胞外结构域产生。将通过流式细胞术分选标记的细胞，分析CPR阳性并在不同厌氧条件下培养。然后对含有生长中的CPR代表的培养试验进行生理分析。将使用荧光和低温透射电子显微镜分析CPR细菌及其共生宿主的共培养物，以解析CPR细菌及其宿主的形态和相互作用。在该项目的合作部分，CPR细菌培养物的上清液将进行第一次代谢组学分析。纯共培养物将最终用于基因组测序和代谢模型的构建。将筛选基因组序列以寻找新的抗体靶标，以优化抗体促进的CPR细菌捕获。综上所述，项目工作流程分为以下三个部分：A部分：CPR细菌的样本采集、处理、细胞标记和分选; B部分：CPR分离株的培养和生理生化分析; C部分：单细胞基因组测序、代谢重建和用于CPR捕获的新抗体的开发将使用新开发的抗体重复该工作流程以捕获，分离和表征更多的CPR细菌，并评估更多CPR分离物的生理学和生物化学。因此，该项目将产生一种新的工具，使CPR细菌可用于实验室培养，并首次揭示这种广泛而神秘的细菌群的生理学。