CLR-seq
Bacterial microbiota located at host barrier sites  harbor species essential for human health, but also species with pathogenic potential. Recent studies have shown that select microbiota species contribute to antibody-based host defense by inducing local and systemic antibody responses. However, the molecular mechanisms underlying the selection of these immunologically-relevant microbiota are still unknown. Barrier-resident antigen-presenting cells (APCs) are critical to initiate local and systemic immune responses upon bacterial invasion. Furthermore, APCs sample the environment during homeostasis by extending their dendrites, which are densely covered with glycan-binding C-type lectin receptors (CLRs), across intact epithelial barriers. Thereby, APCs are ideally positioned and equipped to probe surface-expressed bacterial glycans to induce and sustain local and systemic immunity. To identify the repertoire of microbiota species that could be sampled by APCs, we have developed CLR-sequencing (CLR-seq), combining CLR-based bacterial cell sorting and 16S rRNA gene sequencing. We established and optimized our experimental pipeline using the fluorescently-labeled CLRs langerin (CD207) and the macrophage galactose C-type lectin (MGL, CD301), and fecal microbiota samples from healthy donors. The proportion of langerin- and MGL-positive microbiota populations showed inter-individual variation but shared enrichment of specific genera. Moreover, CLR-enriched genera correlated with IgA-enriched bacterial genera, suggesting a link between glycan-based recognition of bacterial species and the induction of local antibody production. Finally, we validated CLR interaction for several species with high CLR enrichment using flow cytometry. In summary, CLR-seq allows identification of human microbiota species based on CLR-interacting glycans with easy expansion to include additional CLRs or patient microbiota samples.