Glycan profiling of the gut microbiota by Glycan-seq

Abstract

Bacterial glycans modulate the cross talk between the gut microbiota and its host. However, little is known about these glycans because of the lack of appropriate technology to study them. In this study, we applied Glycan-seq technology for glycan profiling of the intact gut microbiota of mice. The evaluation of cultured gram-positive (Deinococcus radiodurans) and gram-negative (Escherichia coli) bacteria showed significantly distinct glycan profiles between these bacteria, which were selected and further analyzed by flow cytometry. The results of flow cytometry agreed well with those obtained by Glycan-seq, indicating that Glycan-seq can be used for bacterial glycan profiling. We thus applied Glycan-seq for comparative glycan profiling of pups and adult mice gut microbiotas. The glycans of the pups and adult microbiotas had significantly distinct glycan profiles, which reflect the different bacterial compositions of pups and adult gut microbiotas based on 16S rRNA gene sequencing.α2-6Sia-binders bound specifically to the pups microbiota. Lectin pull-down followed by 16S rRNA gene sequencing of the pups microbiota identified Lactobacillaceae as the most abundant bacterial family with glycans reacting with α2-6Sia-binders. The Glycan-seq system can reveal the glycan profile of the intact bacterial gut microbiota.

Glycan Profiling by Sequencing to Uncover Multicellular Communication: Launching Glycobiology in Single Cells and Microbiomes

“…To confirm the Glycan-seq data, lectins differentially detected in the cultured bacteria were subjected to flow cytometry analysis, the gold standard for cellular lectin staining. The results indicated that the identified lectins are bound to the bacteria, which is in agreement with the Glycan-seq data and suggested that Glycan-seq data may be applied to bacterial cell surface glycan analysis ( Oinam et al, 2022 ). The specific lectin binding was confirmed using a competition assay with saccharide inhibitors and glycosidase treatment, followed by flow cytometry.…”

“…Each colored bar represents the bacterial family identified. Figures are reprinted from Oinam et al (2022 ) ( Oinam et al, 2022 ).…”

“…For this purpose, the Glycan-seq technique described earlier was modified and implemented for the analysis of bacterial surface glycans. We first evaluated the applicability of Glycan-seq to bacterial glycan profiling in cultured bacteria: Gram-positive ( Deinococcus radiodurans ) vs. Gram-negative ( Escherichia coli ) bacteria ( Oinam et al, 2022 ). After incubation of bacteria with DNA-barcoded lectins, the barcodes were released from the lectins bound to the bacteria by UV irradiation.…”

“…After incubation of bacteria with DNA-barcoded lectins, the barcodes were released from the lectins bound to the bacteria by UV irradiation. The recovered DNA barcodes were amplified by PCR, and the number of barcodes derived from each lectin was counted by a next-generation sequencer, which corresponds to the resulting bacterial glycan profiles ( Oinam et al, 2022 ). The Glycan-seq analysis revealed that the glycans were different between Gram-positive and Gram-negative bacteria.…”

“…After confirming the applicability of glycan analysis to cultured bacteria, Glycan-seq was used to analyze the gut microbiota along with genomic profiling of the bulk gut microbiota from pups (14–20 days old) and adult (12-month old) mice ( Figure 2B ) ( Oinam et al, 2022 ). Glycan analysis revealed that the glycans of mouse pups and adult mice were different ( Figure 2B ) and lectins identifying sialylated glycans were higher in pups ( Figure 2C ).…”