肠道微生物群在维持全身和肠道稳态​​方面发挥着重要作用，但也会影响炎症性肠病（IBD）和癌症等疾病。肠道内的某些细菌种类可以长期激活免疫系统，导致低度肠道炎症。结果，浆细胞产生高水平的分泌性抗原特异性免疫球蛋白 A (IgA)，其包裹着免疫刺激细菌。这种针对肠道细菌的 IgA 免疫反应可能与维持体内平衡和健康以及疾病有关。解开这种二分法并识别免疫刺激细菌对于了解肠道微生物群与免疫系统之间的关系及其在健康和疾病中的作用至关重要。 IgA-SEQ 技术已成功从粪便中鉴定出具有免疫刺激性的 IgA 包被细菌。但原有技术耗时长，下游应用受到限制。在这项研究中，我们的目标是开发一种下一代、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。我们表明，在各种复杂性设置下，从简单的细菌混合物到人类粪便样本，我们的基于磁性 96 孔板的 ng-IgA-SEQ 方案能够以高通量和省时的方式高效地分选和识别 IgA 包被的细菌。此外，我们对不同的 IgA-SEQ 方案进行了比较分析，强调原始基于 FACS 的 IgA-SEQ 方法由于分选细菌的产量低而忽略了 IgA 包被细菌的某些细微差别。此外，基于磁性的 ng-IgA-SEQ 允许新颖的下游应用。首先，作为概念验证，我们对 10 份人类粪便样本进行了宏基因组鸟枪法测序，以鉴定 IgA 包被的细菌菌株以及相关途径和 CAZymes。其次，我们通过在厌氧条件下执行分离方案，成功分离和培养了 IgA 包被细菌。我们基于磁性 96 孔板的高通量下一代 IgA-SEQ 技术有效地从粪便中鉴定了大量 IgA 包被细菌。样品。这为分析大型群体以及新颖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细菌在健康和疾病中的作用至关重要。视频摘要。© 2024。作者。

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.© 2024. The Author(s).