Bifidobacterium infantis utilizes N-acetylglucosamine-containing human milk oligosaccharides as a nitrogen source

Abstract

Bifidobacterium longum subsp. infantis (B. infantis) utilizes oligosaccharides secreted in human milk as a carbohydrate source. These human milk oligosaccharides (HMOs) integrate the nitrogenous residue N-acetylglucosamine (NAG), although HMO nitrogen utilization has not been described to date. Herein, we characterize the B. infantis nitrogen utilization phenotype on two NAG-containing HMO species, LNT and LNnT. This was characterized through in vitro growth kinetics, incorporation of isotopically labeled NAG nitrogen into the proteome, as well as modulation of intracellular 2-oxoglutarate levels while utilizing HMO nitrogen. Further support is provided by comparative transcriptomics and proteomics that identified global regulatory networks deployed during HMO nitrogen utilization. The aggregate data demonstrate that B. infantis strains utilize HMO nitrogen with the potential to significantly impact fundamental and clinical studies, as well as enable applications.

Keywords: 2-oxoglutarate; bifidobacteria; human milk oligosaccharides; microbiota; nitrogen metabolism.

Figure 1.

Terminal asymptotic optical density at 600_nm while B. infantis utilizes LNT or LNnT as a nitrogen source. Data were grouped into three panels according to carbohydrate conditions: (A) lactose, (B) LNT, and (C) LNnT. Adjusted P < 0.05 was designated significant.

Figure 2.

B. infantis differentially expressed proteins and genes while utilizing NAG as a nitrogen source. Differentially expressed genes during NAG utilization consistently within the transcriptome are colored in red. (A) differentially regulated proteins (DRPs) during NAG or complex nitrogen utilization. (B) differentially expressed proteins during NAG or L-cysteine utilization. Upregulated DRPs are colored in blue, and downregulated DRPs are colored in yellow. Asterisks indicate that the protein contain a ¹⁵N label. Log2 fold change (Log2fc > 1.5, p < 0.1 FDR corrected) values are indicated on x-axis.

Figure 3.

B. infantis intracellular 2-OG concentrations while B. infantis utilizes LNT, LNnT, or peptone nitrogen. Data are grouped into three panels according to carbohydrates fermented in the presence of a defined nitrogen: (A) lactose, (B) LNT, (C) LNnT. Adjusted P < 0.05 was designated significant.

Figure 4.

Model of B. infantis HMO nitrogen utilization. Nitrogen metabolism is shaded in yellow, carbohydrate metabolism in green, and peptidoglycan synthesis pathways in purple. Solid arrows are for one-step reactions and dashed arrows for multi-step reactions. The genes involved in the pathways are designated by numbers (1–25) in dark gray circles and listed in Table S5. LNT, lacto-N-tetraose; LNnT, lacto-N-neotetraose; NAG or GlcNAc, N-acetyl glucosamine; GlcNAc-6-P, N-acetyl glucosamine-6-phosphate; GlcNAc-1-P, N-acetyl glucosamine-1-phosphate; GlcN-6-P, glucosamine-6-phosphate; GlcN-1-P, glucosamine-1-phosphate; Fru-6-P, fructose-6-phosphate; 2-OG, 2-oxoglutarate; Glu, glutamate; Gln, glutamine; UDP-GlcNAc, uridine diphosphate-N-acetyl glucosamine; UDP-MurNAc, uridine diphosphate-N-acetyl muramic acid.