The metabolic profile of Bifidobacterium dentium reflects its status as a human gut commensal

Abstract

Background: Bifidobacteria are commensal microbes of the mammalian gastrointestinal tract. In this study, we aimed to identify the intestinal colonization mechanisms and key metabolic pathways implemented by Bifidobacterium dentium.

Results: B. dentium displayed acid resistance, with high viability over a pH range from 4 to 7; findings that correlated to the expression of Na+/H+ antiporters within the B. dentium genome. B. dentium was found to adhere to human MUC2+ mucus and harbor mucin-binding proteins. Using microbial phenotyping microarrays and fully-defined media, we demonstrated that in the absence of glucose, B. dentium could metabolize a variety of nutrient sources. Many of these nutrient sources were plant-based, suggesting that B. dentium can consume dietary substances. In contrast to other bifidobacteria, B. dentium was largely unable to grow on compounds found in human mucus; a finding that was supported by its glycosyl hydrolase (GH) profile. Of the proteins identified in B. dentium by proteomic analysis, a large cohort of proteins were associated with diverse metabolic pathways, indicating metabolic plasticity which supports colonization of the dynamic gastrointestinal environment.

Conclusions: Taken together, we conclude that B. dentium is well adapted for commensalism in the gastrointestinal tract.

Keywords: Acid stress; Bifidobacteria; Carbohydrates; Commensal; Glycans; Intestine; Metabolism.

Fig. 1

Bifidobacterium dentium is resistant to acid stress. a Representative images of live/dead staining of B. dentium ATCC 27678 after 2 h incubation in media at pH 7, 6, 5, 4, and 3. Inserts at high magnification highlight the abundance of live (green) and dead (red) B. dentium at pH 7 and pH 3 (scale bar = 50 μm). b Quantitation of live/dead cell staining on a fluorescent plate reader. c Intracellular pH analysis of B. dentium with pHrodo Red pH sensitive dye. Variance in intracellular pH is reflected by the change in relative fluorescence units (RFU), at various extracellular pH values over 50 min. d Calculated final intracellular pH values at t = 50 min. All data are presented as mean ± stdev

Fig. 2

B. dentium adheres to mucus-producing human intestinal epithelial cells. a Representative immunofluorescence images of B. dentium ATCC 27678 (yellow) co-localization with MUC2 (blue) in mucin-producing human HT29-MTX colonic cells after 1 h incubation (scale bar = 50 μm). b Scanning electron micrograph of B. dentium and HT29-MX cells after 1 h incubation (scale bar = 5 μm)

Fig. 3

B. dentium grows on select sugars in the absence of glucose. B. dentium ATCC 27678 was grown anaerobically at 37 °C in Biolog plates with a fully-defined media (LDM4) preparation that lacked glucose. Growth was monitored over 16 h by plate reader in plate containing a hexoses, b pentoses, c ketoses, d disaccharides, e trisaccharides, f sugar alcohols, g deoxy sugars and h amino sugars. i For visualization, heat maps were generated for all sugars at time 0, 8.3 and 16.0 h. All data are presented as mean ± stdev

Fig. 4

The B. dentium ATCC 27678 genome contains mulitple glycosyl hydrolase (GH) genes. The B. dentium ATCC 27678 genome was found to harbor 88 GH-related genes, encoding for 25 different GH families

Fig. 5

B. dentium yields minimal growth on amino acids and amino acid derivatives in LDM4 preparations prepared without glucose. B. dentium ATCC 27678 was grown anaerobically at 37 °C in Biolog plates with a fully-defined media (LDM4) preparation that lacked glucose. Growth was monitored over 16 h by plate reader in plate containing (a) 33 different amino acids. b For visualization, heat maps were generated for all amino acids at time 0, 8.3 and 16.0 h. All data are presented as mean ± stdev

Fig. 6

B. dentium does not grow on glycosides, nucleosides, polymers, polysaccharides or polysorbates in the absence of glucose, with the exception of amygdalin, arbutin and salicin. B. dentium ATCC 27678 was grown anaerobically at 37 °C in Biolog plates with a fully-defined media (LDM4) preparation that lacked glucose. Growth was monitored over 16 h by plate reader in plate containing a glycosides, b nucleosides. Heat maps were generated for c glycosides and d nucleosides at time 0, 8.3 and 16 h. B. dentium growth was also monitored with e polymers, f polysaccharides, and g polysorbates. h For visualization, heat maps were generated for polymers, polysaccharides and polysorbates at time 0, 8.3 and 16.0 h. All data are presented as mean ± stdev

Fig. 7

B. dentium has minimal growth on organic acids without glucose. B. dentium ATCC 27678 was grown anaerobically at 37 °C in Biolog plates with a fully-defined media (LDM4) preparation that lacked glucose. Growth was monitored over 16 h by plate reader in plate containing 59 different organic acids. Acids were separated into groups: a 12 acids, b 9, c 9, d 8 and e 21 acids. f Heat maps were generated for organic acids at time 0, 8.3 and 16.0 h. All data are presented as mean ± stdev

Fig. 8

Pathway analysis B. dentium by proteomic analysis. B. dentium ATCC 27678 were examined using high-resolution liquid chromatography-tandem mass spectrometry based proteomics and 319 proteins were identified from B. dentium. The functional classifications of these proteins are illustrated in the pie chart above

Fig. 9

Proposed model for B. dentium intestinal colonization. Our data suggest that B. dentium is acid resistant, adheres to the intestinal mucus layer and consumes a variety of dietary sources. We speculate that these features contribute to the ability of B. dentium to colonize the intestine