新一代IgA-SEQ实现高通量、厌氧和宏基因组水平的IgA包被细菌评估

肠道微生物群在维持系统性和肠道稳态中起着重要作用，但也可影响如炎症性肠病（IBD）和癌症等疾病。肠道中的某些细菌种类能够持续激活免疫系统，导致低度肠道炎症。因此，血浆细胞产生高水平的分泌性抗原特异性免疫球蛋白A（IgA），并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稳态与健康以及疾病有关。揭示这一二元性并识别免疫刺激性细菌，对于理解肠道微生物群与免疫系统的关系及其在健康与疾病中的作用至关重要。IgA-SEQ技术已成功识别来自粪便样本的免疫刺激性、IgA包被细菌。然而，原始技术耗时较长，且下游应用有限。本研究旨在开发新一代高通量磁性分选方法（ng-IgA-SEQ），以克服原始IgA-SEQ方案的限制。我们在各种复杂度环境下（从简单细菌混合物到人类粪便样本）展示了，基于磁性96孔板的ng-IgA-SEQ方案在高效分选和识别IgA包被细菌方面表现出色，具有高通量和节省时间的优势。此外，我们对不同IgA-SEQ方案进行了比较分析，强调原始基于流式细胞仪（FACS）的方法因分选细菌产量低，可能会忽略某些细菌的特征。磁性ng-IgA-SEQ还支持多种新型下游应用。首先，作为概念验证，我们对10个人体粪便样本进行了宏基因组鸟枪法测序，识别了IgA包被细菌菌株及其相关通路和CAZymes。其次，我们在厌氧条件下成功分离和培养了IgA包被细菌。基于磁性96孔板的高通量新一代IgA-SEQ技术高效识别大量粪便样本中的IgA包被细菌，为大规模队列分析以及包括鸟枪宏基因组测序、培养组学和多种功能性分析提供了基础。这些下游应用对于揭示免疫刺激性细菌在健康与疾病中的作用至关重要。视频摘要。

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.