Medical Microbiology Department, UMC Utrecht, Utrecht, The Netherlands.
Department of Gastroenterology and Hepatology, UMC Utrecht, Utrecht, The Netherlands.
Microbiome. 2024 Oct 21;12(1):211. doi: 10.1186/s40168-024-01923-9.
The intestinal microbiota plays a significant role in maintaining systemic and intestinal homeostasis, but can also influence diseases such as inflammatory bowel disease (IBD) and cancer. Certain bacterial species within the intestinal tract can chronically activate the immune system, leading to low-grade intestinal inflammation. As a result, plasma cells produce high levels of secretory antigen-specific immunoglobulin A (IgA), which coats the immunostimulatory bacteria. This IgA immune response against intestinal bacteria may be associated with the maintenance of homeostasis and health, as well as disease. Unraveling this dichotomy and identifying the immunostimulatory bacteria is crucial for understanding the relationship between the intestinal microbiota and the immune system, and their role in health and disease. IgA-SEQ technology has successfully identified immunostimulatory, IgA-coated bacteria from fecal material. However, the original technology is time-consuming and has limited downstream applications. In this study, we aimed to develop a next-generation, high-throughput, magnet-based sorting approach (ng-IgA-SEQ) to overcome the limitations of the original IgA-SEQ protocol.
We show, in various settings of complexity ranging from simple bacterial mixtures to human fecal samples, that our magnetic 96-well plate-based ng-IgA-SEQ protocol is highly efficient at sorting and identifying IgA-coated bacteria in a high-throughput and time efficient manner. Furthermore, we performed a comparative analysis between different IgA-SEQ protocols, highlighting that the original FACS-based IgA-SEQ approach overlooks certain nuances of IgA-coated bacteria, due to the low yield of sorted bacteria. Additionally, magnetic-based ng-IgA-SEQ allows for novel downstream applications. Firstly, as a proof-of-concept, we performed metagenomic shotgun sequencing on 10 human fecal samples to identify IgA-coated bacterial strains and associated pathways and CAZymes. Secondly, we successfully isolated and cultured IgA-coated bacteria by performing the isolation protocol under anaerobic conditions.
Our magnetic 96-well plate-based high-throughput next-generation IgA-SEQ technology efficiently identifies a great number of IgA-coated bacteria from fecal samples. This paves the way for analyzing large cohorts as well as novel downstream applications, including shotgun metagenomic sequencing, culturomics, and various functional assays. These downstream applications are essential to unravel the role of immunostimulatory bacteria in health and disease. Video Abstract.
肠道微生物群在维持全身和肠道内环境稳定方面起着重要作用，但也会影响炎症性肠病（IBD）和癌症等疾病。肠道内的某些细菌物种可以慢性激活免疫系统，导致低度肠道炎症。结果，浆细胞产生高水平的分泌型抗原特异性免疫球蛋白 A（IgA），该免疫球蛋白 A 覆盖免疫刺激性细菌。这种针对肠道细菌的 IgA 免疫反应可能与维持内环境稳定和健康以及疾病有关。揭示这种二分法并确定免疫刺激性细菌对于理解肠道微生物群与免疫系统之间的关系以及它们在健康和疾病中的作用至关重要。IgA-SEQ 技术已成功地从粪便中鉴定出免疫刺激性、IgA 包被的细菌。然而，原始技术耗时且下游应用有限。在这项研究中，我们旨在开发一种下一代、高通量、基于磁体的分选方法（ng-IgA-SEQ），以克服原始 IgA-SEQ 方案的局限性。
我们展示了，在从简单的细菌混合物到人类粪便样本的各种复杂环境中，我们的基于磁性 96 孔板的 ng-IgA-SEQ 方案以高通量和高效的方式分选和鉴定 IgA 包被细菌的效率非常高。此外，我们对不同的 IgA-SEQ 方案进行了比较分析，突出了由于分选细菌的产量低，原始基于 FACS 的 IgA-SEQ 方法会忽略 IgA 包被细菌的某些细微差别。此外，基于磁体的 ng-IgA-SEQ 允许进行新的下游应用。首先，作为概念验证，我们对 10 个人类粪便样本进行了宏基因组鸟枪法测序，以鉴定 IgA 包被细菌菌株及其相关途径和 CAZymes。其次，我们通过在厌氧条件下进行分离方案成功地分离和培养了 IgA 包被细菌。
我们的基于磁性 96 孔板的高通量下一代 IgA-SEQ 技术能够从粪便样本中高效地鉴定出大量 IgA 包被细菌。这为分析大量队列以及新的下游应用铺平了道路，包括鸟枪法宏基因组测序、培养组学和各种功能测定。这些下游应用对于揭示免疫刺激性细菌在健康和疾病中的作用至关重要。视频摘要。
