GH20 and GH84 ß-N-acetylglucosaminidases with different linkage specificities underpin mucin O-glycan breakdown capability of Bifidobacterium bifidum.

Intestinal mucous layers mediate symbiosis and dysbiosis of host-microbe interactions. These interactions are influenced by the mucin O-glycan degrading ability of several gut microbes. The identities and prevalence of many glycoside hydrolases (GHs) involved in microbial mucin O-glycan breakdown have been previously reported; however, the exact mechanisms and extent to which these GHs are dedicated to mucin O-glycan degradation pathways warrant further research. Here, using Bifidobacterium bifidum as a model mucinolytic bacterium, we revealed that two ß-N-acetylglucosaminidases belonging to the GH20 (BbhI) and GH84 (BbhIV) families play important roles in mucin O-glycan degradation. Using substrate specificity analysis of natural oligosaccharides and O-glycomic analysis of porcine gastric mucin (PGM) incubated with purified enzymes or B. bifidum carrying bbhI and/or bbhIV mutations, we showed that BbhI and BbhIV are highly specific for ß-(1→3)- and ß-(1→6)-GlcNAc linkages of mucin core structures, respectively. Interestingly, we found that efficient hydrolysis of the ß-(1→3)-linkage by BbhI of the mucin core 4 structure [GlcNAcß1-3(GlcNAcß1-6)GalNAcα-O-Thr] required prior removal of the ß-(1→6)-GlcNAc linkage by BbhIV. Consistent with this, inactivation of bbhIV markedly decreased the ability of B. bifidum to release GlcNAc from PGM. When combined with a bbhI mutation, we observed that the growth of the strain on PGM was reduced. Finally, phylogenetic analysis suggests that GH84 members may have gained diversified functions through microbe-microbe and host-microbe horizontal gene transfer events. Taken together, these data strongly suggest the involvement of GH84 family members in host glycan breakdown.

Bifidobacterium bifidum strains synergize with immune checkpoint inhibitors to reduce tumour burden in mice.

The gut microbiome can influence the development of tumours and the efficacy of cancer therapeutics1-5; however, the multi-omics characteristics of antitumour bacterial strains have not been fully elucidated. In this study, we integrated metagenomics, genomics and transcriptomics of bacteria, and analyses of mouse intestinal transcriptome and serum metabolome data to reveal an additional mechanism by which bacteria determine the efficacy of cancer therapeutics. In gut microbiome analyses of 96 samples from patients with non-small-cell lung cancer, Bifidobacterium bifidum was abundant in patients responsive to therapy. However, when we treated syngeneic mouse tumours with commercial strains of B. bifidum to establish relevance for potential therapeutic uses, only specific B. bifidum strains reduced tumour burden synergistically with PD-1 blockade or oxaliplatin treatment by eliciting an antitumour host immune response. In mice, these strains induced tuning of the immunological background by potentiating the production of interferon-γ, probably through the enhanced biosynthesis of immune-stimulating molecules and metabolites.

Human milk and mucosa-associated disaccharides impact on cultured infant fecal microbiota.

Human milk oligosaccharides (HMOs) are a mixture of structurally diverse carbohydrates that contribute to shape a healthy gut microbiota composition. The great diversity of the HMOs structures does not allow the attribution of specific prebiotic characteristics to single milk oligosaccharides. We analyze here the utilization of four disaccharides, lacto-N-biose (LNB), galacto-N-biose (GNB), fucosyl-α1,3-GlcNAc (3FN) and fucosyl-α1,6-GlcNAc (6FN), that form part of HMOs and glycoprotein structures, by the infant fecal microbiota. LNB significantly increased the total levels of bifidobacteria and the species Bifidobacterium breve and Bifidobacterium bifidum. The Lactobacillus genus levels were increased by 3FN fermentation and B. breve by GNB and 3FN. There was a significant reduction of Blautia coccoides group with LNB and 3FN. In addition, 6FN significantly reduced the levels of Enterobacteriaceae family members. Significantly higher concentrations of lactate, formate and acetate were produced in cultures containing either LNB or GNB in comparison with control cultures. Additionally, after fermentation of the oligosaccharides by the fecal microbiota, several Bifidobacterium strains were isolated and identified. The results presented here indicated that each, LNB, GNB and 3FN disaccharide, might have a specific beneficial effect in the infant gut microbiota and they are potential prebiotics for application in infant foods.

Probiotics modulate gut microbiota and improve insulin sensitivity in DIO mice.

Obesity and type 2 diabetes are characterized by subclinical inflammatory process. Changes in composition or modulation of the gut microbiota may play an important role in the obesity-associated inflammatory process. In the current study, we evaluated the effects of probiotics (Lactobacillus rhamnosus, L. acidophilus and Bifidobacterium bifidumi) on gut microbiota, changes in permeability, and insulin sensitivity and signaling in high-fat diet and control animals. More importantly, we investigated the effects of these gut modulations on hypothalamic control of food intake, and insulin and leptin signaling. Swiss mice were submitted to a high-fat diet (HFD) with probiotics or pair-feeding for 5 weeks. Metagenome analyses were performed on DNA samples from mouse feces. Blood was drawn to determine levels of glucose, insulin, LPS, cytokines and GLP-1. Liver, muscle, ileum and hypothalamus tissue proteins were analyzed by Western blotting and real-time polymerase chain reaction. In addition, liver and adipose tissues were analyzed using histology and immunohistochemistry. The HFD induced huge alterations in gut microbiota accompanied by increased intestinal permeability, LPS translocation and systemic low-grade inflammation, resulting in decreased glucose tolerance and hyperphagic behavior. All these obesity-related features were reversed by changes in the gut microbiota profile induced by probiotics. Probiotics also induced an improvement in hypothalamic insulin and leptin resistance. Our data demonstrate that the intestinal microbiome is a key modulator of inflammatory and metabolic pathways in both peripheral and central tissues. These findings shed light on probiotics as an important tool to prevent and treat patients with obesity and insulin resistance.

Review on Bifidobacterium bifidum BGN4: Functionality and Nutraceutical Applications as a Probiotic Microorganism.

Bifidobacterium bifidum BGN4 is a probiotic strain that has been used as a major ingredient to produce nutraceutical products and as a dairy starter since 2000. The various bio-functional effects and potential for industrial application of B. bifidum BGN4 has been characterized and proven by in vitro (i.e., phytochemical bio-catalysis, cell adhesion and anti-carcinogenic effects on cell lines, and immunomodulatory effects on immune cells), in vivo (i.e., suppressed allergic responses in mouse model and anti-inflammatory bowel disease), and clinical studies (eczema in infants and adults with irritable bowel syndrome). Recently, the investigation of the genome sequencing was finished and this data potentially clarifies the biochemical characteristics of B. bifidum BGN4 that possibly illustrate its nutraceutical functionality. However, further systematic research should be continued to gain insight for academic and industrial applications so that the use of B. bifidum BGN4 could be expanded to result in greater benefit. This review deals with multiple studies on B. bifidum BGN4 to offer a greater understanding as a probiotic microorganism available in functional food ingredients. In particular, this work considers the potential for commercial application, physiological characterization and exploitation of B. bifidum BGN4 as a whole.