GH20 and GH84 β-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 β-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 β-(1→3)- and β-(1→6)-GlcNAc linkages of mucin core structures, respectively. Interestingly, we found that efficient hydrolysis of the β-(1→3)-linkage by BbhI of the mucin core 4 structure [GlcNAcβ1-3(GlcNAcβ1-6)GalNAcα-O-Thr] required prior removal of the β-(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

Human Milk Oligosaccharide Utilization in Intestinal Bifidobacteria Is Governed by Global Transcriptional Regulator NagR

Bifidobacterium longum subsp. infantis is a prevalent beneficial bacterium that colonizes the human neonatal gut and is uniquely adapted to efficiently use human milk oligosaccharides (HMOs) as a carbon and energy source. Multiple studies have focused on characterizing the elements of HMO utilization machinery in B. longum subsp. infantis; however, the regulatory mechanisms governing the expression of these catabolic pathways remain poorly understood. A bioinformatic regulon reconstruction approach used in this study implicated NagR, a transcription factor from the ROK family, as a negative global regulator of gene clusters encoding lacto-N-biose/galacto-N-biose (LNB/GNB), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT) utilization pathways in B. longum subsp. infantis. This conjecture was corroborated by transcriptome profiling upon nagR genetic inactivation and experimental assessment of binding of recombinant NagR to predicted DNA operators. The latter approach also implicated N-acetylglucosamine (GlcNAc), a universal intermediate of LNT and LNnT catabolism, and its phosphorylated derivatives as plausible NagR transcriptional effectors. Reconstruction of NagR regulons in various Bifidobacterium lineages revealed multiple potential regulon expansion events, suggesting evolution from a local regulator of GlcNAc catabolism in ancestral bifidobacteria to a global regulator controlling the utilization of mixtures of GlcNAc-containing host glycans in B. longum subsp. infantis and Bifidobacterium bifidum

A simple method that enhances minority species detection in the microbiota: 16S metagenome-DRIP (Deeper Resolution using an Inhibitory Primer)

Aim: 16S rRNA gene-based microbiota analyses (16S metagenomes) using next-generation sequencing (NGS) technologies are widely used to examine the microbial community composition in environmental samples. However, the sequencing capacity of NGS is sometimes insufficient to cover the whole microbial community, especially when analyzing soil and fecal microbiotas. This limitation may have hampered the detection of minority species that potentially affect microbiota formation and structure. Methods: We developed a simple method, termed 16S metagenome-DRIP (Deeper Resolution using an Inhibitory Primer), that not only enhances minority species detection but also increases the accuracy of their abundance estimation. The method relies on the inhibition of normal amplicon formation of the 16S rRNA gene of a target major (abundant) species during the first PCR step. The addition of a biotinylated primer that is complementary to the variable sequence of the V3-V4 region of the target species inhibits a normal amplification process to form an aberrant short amplicon. The fragment is then captured by streptavidin beads for removal from the reaction mixture, and the resulting mixture is utilized for the second PCR with barcode-tag primers. Thus, this method only requires two additional experimental procedures to the conventional 16S metagenome analysis. A proof-of-concept experiment was first conducted using a mock sample consisting of the genomes of 14 bacterial species. Then, the method was applied to infant fecal samples using a Bifidobacterium-specific inhibitory primer (n = 11). Results: As a result, the reads assigned to the family Bifidobacteriaceae decreased on average from 16, 657 to 1718 per sample without affecting the total read counts (36, 073 and 34, 778 per sample for the conventional and DRIP methods, respectively). Furthermore, the minority species detection rate increased with neither affecting Bray-Curtis dissimilarity calculated by omitting the target Bifidobacterium species (median: 0.049) nor changing the relative abundances of the non-target species. While 115 amplicon sequence variants (ASVs) were unique to the conventional method, 208 ASVs were uniquely detected for the DRIP method. Moreover, the abundance estimation for minority species became more accurate, as revealed thorough comparison with the results of quantitative PCR analysis. Conclusion: The 16S metagenome-DRIP method serves as a useful technique to grasp a deeper and more accurate microbiota composition when combined with conventional 16S metagenome analysis methods

Bifidobacterium response to lactulose ingestion in the gut relies on a solute-binding protein-dependent ABC transporter

ビフィズス菌がラクチュロースを利用する仕組みを解明 --ビフィズス菌の増殖作用の予測への活用も--. 京都大学プレスリリース. 2021-05-24.This study aims to understand the mechanistic basis underlying the response of Bifidobacterium to lactulose ingestion in guts of healthy Japanese subjects, with specific focus on a lactulose transporter. An in vitro assay using mutant strains of Bifidobacterium longum subsp. longum 105-A shows that a solute-binding protein with locus tag number BL105A_0502 (termed LT-SBP) is primarily involved in lactulose uptake. By quantifying faecal abundance of LT-SBP orthologues, which is defined by phylogenetic analysis, we find that subjects with 10⁷ to 10⁹ copies of the genes per gram of faeces before lactulose ingestion show a marked increase in Bifidobacterium after ingestion, suggesting the presence of thresholds between responders and non-responders to lactulose. These results help predict the prebiotics-responder and non-responder status and provide an insight into clinical interventions that test the efficacy of prebiotics

Priority effects shape the structure of infant-type Bifidobacterium communities on human milk oligosaccharides

母乳栄養児の腸内におけるビフィズス菌コミュニティー形成には先住効果が大きな影響を及ぼす --ヒトミルクオリゴ糖利用能力の低いビフィズス菌B. breveが優勢となる仕組み--. 京都大学プレスリリース. 2022-07-27.Bifidobacteria are among the first colonizers of the infant gut, and human milk oligosaccharides (HMOs) in breastmilk are instrumental for the formation of a bifidobacteria-rich microbiota. However, little is known about the assembly of bifidobacterial communities. Here, by applying assembly theory to a community of four representative infant-gut associated Bifidobacterium species that employ varied strategies for HMO consumption, we show that arrival order and sugar consumption phenotypes significantly affected community formation. Bifidobacterium bifidum and Bifidobacterium longum subsp. infantis, two avid HMO consumers, dominate through inhibitory priority effects. On the other hand, Bifidobacterium breve, a species with limited HMO-utilization ability, can benefit from facilitative priority effects and dominates by utilizing fucose, an HMO degradant not utilized by the other bifidobacterial species. Analysis of publicly available breastfed infant faecal metagenome data showed that the observed trends for B. breve were consistent with our in vitro data, suggesting that priority effects may have contributed to its dominance. Our study highlights the importance and history dependency of initial community assembly and its implications for the maturation trajectory of the infant gut microbiota

<i>N</i>-Carbamoylputrescine Amidohydrolase of <i>Bacteroides thetaiotaomicron</i>, a Dominant Species of the Human Gut Microbiota

Polyamines are bioactive amines that play a variety of roles, such as promoting cell proliferation and protein synthesis, and the intestinal lumen contains up to several mM polyamines derived from the gut microbiota. In the present study, we conducted genetic and biochemical analyses of the polyamine biosynthetic enzyme N-carbamoylputrescine amidohydrolase (NCPAH) that converts N-carbamoylputrescine to putrescine, a precursor of spermidine in Bacteroides thetaiotaomicron, which is one of the most dominant species in the human gut microbiota. First, ncpah gene deletion and complemented strains were generated, and the intracellular polyamines of these strains cultured in a polyamine-free minimal medium were analyzed using high-performance liquid chromatography. The results showed that spermidine detected in the parental and complemented strains was depleted in the gene deletion strain. Next, purified NCPAH-(His)6 was analyzed for enzymatic activity and found to be capable of converting N-carbamoylputrescine to putrescine, with a Michaelis constant (Km) and turnover number (kcat) of 730 µM and 0.8 s−1, respectively. Furthermore, the NCPAH activity was strongly (>80%) inhibited by agmatine and spermidine, and moderately (≈50%) inhibited by putrescine. This feedback inhibition regulates the reaction catalyzed by NCPAH and may play a role in intracellular polyamine homeostasis in B. thetaiotaomicron

Ecological and molecular perspectives on responders and non-responders to probiotics and prebiotics

Bifidobacteria are widely used as a probiotic for their health-promoting effects. To promote their growth, bifidogenic prebiotics, including human milk oligosaccharides (HMOs), have been added to supplements and infant formula. However, the efficacy of both probiotic and prebiotic interventions is often debated, as clinical responses vary significantly by case. Here, we review clinical studies that aimed to proliferate human-residential Bifidobacterium (HRB) strains in the gut, and we highlight the difference between responders and non-responders to such interventions through an ecological, niche-based perspective and an examination of the prevalence of genes responsible for prebiotic assimilation in HRB genomes. We discuss the criteria necessary to better evaluate the efficacy of probiotic and prebiotic interventions and the recent therapeutic potential shown by synbiotics