Release and utilization of N-acetyl-D-glucosamine from human milk oligosaccharides by Bifidobacterium longum subsp. infantis - PubMed (original) (raw)

Release and utilization of N-acetyl-D-glucosamine from human milk oligosaccharides by Bifidobacterium longum subsp. infantis

Daniel Garrido et al. Anaerobe. 2012 Aug.

Abstract

Human milk contains high amounts of complex oligosaccharides, which can be utilized especially by Bifidobacterium species in the infant gut as a carbon and energy source. N-acetyl-D-glucosamine is a building block of these oligosaccharides, and molecular details on the release and utilization of this monosaccharide are not fully understood. In this work we have studied some of the enzymatic properties of three N-acetyl-β-D-hexosaminidases encoded by the genome of the intestinal isolate Bifidobacterium longum subsp. infantis ATCC 15697 and the gene expression of the corresponding genes during bacterial growth on human milk oligosaccharides. These enzymes belong to the glycosyl hydrolase family 20, with several homologs in bifidobacteria. Their optimum pH was 5.0 and optimum temperature was 37 °C. The three enzymes were active on the GlcNAcβ1-3 linkage found in lacto-N-tetraose, the most abundant human milk oligosaccharide. Blon_0459 and Blon_0732, but not Blon_2355, cleaved branched GlcNAcβ1-6 linkages found in lacto-N-hexaose, another oligosaccharide abundant in breast milk. Bifidobacterium infantis N-acetyl-β-D-hexosaminidases were induced during early growth in vitro on human milk oligosaccharides, and also during growth on lacto-N-tetraose or lacto-N-neotetraose. The up-regulation of enzymes that convert this monosaccharide into UDP-N-acetylglucosamine by human milk oligosaccharides suggested that this activated sugar is used in peptidoglycan biosynthesis. These results emphasize the complexity of human milk oligosaccharide consumption by this infant intestinal isolate, and provide new clues into this process.

Copyright © 2012 Elsevier Ltd. All rights reserved.

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Figures

Fig. 1.

Fig. 1.

Thin layer chromatography of co-incubations of B. infantis N-acetyl-β-D-hexosaminidases with LNT or LNH after treatment with β-galactosidases. Structures are illustrated below the figure. Lanes 1–8 and 26–28: standards (as indicated in the figure); lane 9: LNT with specific β1-3 galactosidase; lanes 10–12: LNT with a β1-3 galactosidase and Blon_0459, Blon_0732 or Blon_2355. Lane 13: LNH; lanes 14–16: LNH with either a β1-3, a β1-4 or both specific galactosidases; lanes 17–19: LNH with a β1-3 galactosidase and Blon_0459, Blon_0732 and Blon_2355, respectively; lanes 20–22: LNH with a β1-4 galactosidase and either Blon_0459, Blon_0732 and Blon_2355; lanes 23–25: LNH with both β1-3 and β1-4 galactosidase, as well as either Blon_0459, Blon_0732 and Blon_2355.

Fig. 2.

Fig. 2.

Relative quantification of the gene expression levels of β-hexosaminidase genes at different time points during growth on HMO (A), LNT or LNnT (B). Expression is relative to their levels during growth on lactose. Error bars represent two biological replicates.

Fig. 3.

Fig. 3.

Fold change in gene expression for genes predicted to participate in the metabolism of GlcNAc in B. infantis after exponential growth on the substrates listed in the _x_-axis. Results are normalized to levels on lactose (dashed line), and error bars represent three biological replicates. A: Genes in the LNB/GNB pathway; B: GlcNAc-6-P deacetylase and GlcN-6-P isomerase; C: (results are presented in log scale) a PTS system putative for GlcNAc import and metabolism.

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