Next-Generation IgA-SEQ allows for high-throughout, anaerobic and metagenomic assessment of IgA-coated bacteria
Background The intestinal microbiota plays a significant role in maintaining systemic 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, B cells produce high levels of antigen-specific IgA, which is secreted into the gut, coating the immunostimulatory bacteria that initiated the immune response. The identification of these 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.   Results 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 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 two novel downstream applications. Firstly, as proof-of-concept, we performed metagenomic shotgun sequencing following IgA-SEQ on 10 human fecal samples and identified 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.   Conclusions  Our magnetic, 96-well plate-based high-thoughput next-generation IgA-SEQ protocol efficiently identifies a great number of IgA-coated bacteria from fecal samples. This paves the way for many novel downstream applications, including culturomics, various functional assays, analyzing large datasets and the possibility for shotgun metagenomic sequencing. These downstream applications are essential to unravel the role of immunostimulatory bacteria in health and disease.