Oligosaccharide binding proteins from Bifidobacterium longum subsp. infantis reveal a preference for host glycans

Daniel Garrido, Jae Han Kim, J Bruce German, Helen E Raybould, David A Mills, Daniel Garrido, Jae Han Kim, J Bruce German, Helen E Raybould, David A Mills

Abstract

Bifidobacterium longum subsp. infantis (B. infantis) is a common member of the infant intestinal microbiota, and it has been characterized by its foraging capacity for human milk oligosaccharides (HMO). Its genome sequence revealed an overabundance of the Family 1 of solute binding proteins (F1SBPs), part of ABC transporters and associated with the import of oligosaccharides. In this study we have used the Mammalian Glycan Array to determine the specific affinities of these proteins. This was correlated with binding protein expression induced by different prebiotics including HMO. Half of the F1SBPs in B. infantis were determined to bind mammalian oligosaccharides. Their affinities included different blood group structures and mucin oligosaccharides. Related to HMO, other proteins were specific for oligomers of lacto-N-biose (LNB) and polylactosamines with different degrees of fucosylation. Growth on HMO induced the expression of specific binding proteins that import HMO isomers, but also bind blood group and mucin oligosaccharides, suggesting coregulated transport mechanisms. The prebiotic inulin induced other family 1 binding proteins with affinity for intestinal glycans. Most of the host glycan F1SBPs in B. infantis do not have homologs in other bifidobacteria. Finally, some of these proteins were found to be adherent to intestinal epithelial cells in vitro. In conclusion, this study represents further evidence for the particular adaptations of B. infantis to the infant gut environment, and helps to understand the molecular mechanisms involved in this process.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Oligosaccharides recognized by B. infantis…
Figure 1. Oligosaccharides recognized by B. infantis F1SBPs in the Glycan Array v3.1.
Oligosaccharides are attached to the array using different spacer arms. Sp0: -CH2CH2NH2; Sp8: -CH2CH2CH2NH2; Sp10: -NHCOCH2NH; Sp21: -N(CH3)-O-(CH2)2-NH2. RFU: Relative Fluorescence Units. Yellow circles, galactose; blue circles, glucose; yellow squares, N-Acetyl-galactosamine; blue squares, N-Acetyl-glucosamine; red triangles, fucose; pink diamonds, sialic acid. ND: Not detected.
Figure 2. Gene expression of B. infantis…
Figure 2. Gene expression of B. infantis F1SBPs growing under different prebiotics as the sole carbon source.
Numbers in the x-axis represent each F1SBP locus tag (Blon_). A: 2% HMO; B: 2% Inulin; C: 2% FOS; D: 2% GOS. Colored bars indicated F1SBPs with a change in gene expression higher than two fold; E: relative expression levels for some HMO-induced genes in exponential (blue bars) and stationary phase (white bars); F: relative expression levels for some F1SBPs under growth with GNB (purple bars), LNT (green bars) and LNnT (blue bars) as the only carbon source. Levels for growth on lactose (white bars) are shown as a reference.
Figure 3. Genetic landscapes for Blon_2177 (A),…
Figure 3. Genetic landscapes for Blon_2177 (A), Blon_0883 (B) and Blon_0343 (C), and close related homologs available in the Integrated Microbial Genome database .
Percentages represent similarity of homolog genes to those in the B. infantis ATCC 15697 genome. Locus tags are presented above each gene when available.
Figure 4. Oligosaccharide binding profile proposed for…
Figure 4. Oligosaccharide binding profile proposed for B. infantis cells growing on HMO (A) and inulin (B).
Numbers represent each F1SBP locus tag (Blon_).
Figure 5. Binding of F1SBPs to Caco-2…
Figure 5. Binding of F1SBPs to Caco-2 cells in vitro detected by flow cytometry (A–D) and confocal microcopy (E–F).
A: FITC-streptavidin; B and E: UEA-FITC 0.5 µg/ml; C: Blon_0375-FITC 1 µg/ml; D and F: Blon_2347-FITC 1 µg/ml; G: Blon_2347-FITC 1 µg/ml and DAPI (blue); H: fixed Caco-2 cells coincubated with 1 µg/ml Blon_0375-FITC; I: Caco-2 cells incubated for 10 min with 1 µg/ml Blon_0375-FITC and stained with DAPI.

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