Bacteroides uniformis and its preferred substrate, α-cyclodextrin, enhance endurance exercise performance in mice and human males.

Although gut microbiota has been linked to exercise, whether alterations in the abundance of specific bacteria improve exercise performance remains ambiguous. In a cross-sectional study involving 25 male long-distance runners, we found a correlation between Bacteroides uniformis abundance in feces and the 3000-m race time. In addition, we administered flaxseed lignan or α-cyclodextrin as a test tablet to healthy, active males who regularly exercised in a randomized, double-blind, placebo-controlled study to increase B. uniformis in the gut (UMIN000033748). The results indicated that α-cyclodextrin supplementation improved human endurance exercise performance. Moreover, B. uniformis administration in mice increased swimming time to exhaustion, cecal short-chain fatty acid concentrations, and the gene expression of enzymes associated with gluconeogenesis in the liver while decreasing hepatic glycogen content. These findings indicate that B. uniformis enhances endurance exercise performance, which may be mediated by facilitating hepatic endogenous glucose production.

Administration of ß-lactam antibiotics and delivery method correlate with intestinal abundances of Bifidobacteria and Bacteroides in early infancy, in Japan.

The intestinal microbiome changes dynamically in early infancy. Colonisation by Bifidobacterium and Bacteroides and development of intestinal immunity is interconnected. We performed a prospective observational cohort study to determine the influence of antibiotics taken by the mother immediately before delivery on the intestinal microbiome of 130 healthy Japanese infants. Faecal samples (383) were collected at 1, 3, and 6 months and analysed using next-generation sequencing. Cefazolin was administered before caesarean sections, whereas ampicillin was administered in cases with premature rupture of the membranes and in Group B Streptococcus-positive cases. Bifidobacterium and Bacteroides were dominant (60-70% mean combined occupancy) at all ages. A low abundance of Bifidobacterium was observed in infants exposed to antibiotics at delivery and at 1 and 3 months, with no difference between delivery methods. A lower abundance of Bacteroides was observed after caesarean section than vaginal delivery, irrespective of antibiotic exposure. Additionally, occupancy by Bifidobacterium at 1 and 3 months and by Bacteroides at 3 months differed between infants with and without siblings. All these differences disappeared at 6 months. Infants exposed to intrapartum antibiotics displayed altered Bifidobacterium abundance, whereas abundance of Bacteroides was largely associated with the delivery method. Existence of siblings also significantly influenced the microbiota composition of infants.

Gut microbial composition of elderly women born in the Japanese longevity village Ogimi.

Ogimi is one of Japan's longevity villages and is located in Okinawa Prefecture. In this study, we focused on the elderly women living in the village, classified them into two groups based on whether or not they lived in Ogimi during the first 3 years of their lives, and compared the gut microbiota between the two groups. There were no differences in alpha and beta diversity; however, we found that the elderly women who lived in Ogimi during the first 3 years of their lives had a higher rate of Akkermansia muciniphila colonization in their guts.

Effective bifidogenic growth factors cyclo-Val-Leu and cyclo-Val-Ile produced by Bacillus subtilis C-3102 in the human colonic microbiota model.

Bifidobacterium species are known to fulfill important functions within the human colon. Thus, stimulating the activity of bifidobacteria is important to maintain host health. We revealed that culture supernatants of Bacillus subtilis C-3102 (referred to as C-3102) stimulated the growth of Bifidobacterium species. In this study, we isolated and identified six bifidogenic growth factors, which were cyclo (D-Val-D-Ile), cyclo (L-Val-D-Ile), cyclo (D-Val-L-Ile), cyclo (L-Val-L-Ile), cyclo (D-Val-L-Leu) and cyclo (L-Val-L-Leu). These six cyclic dipeptides increased the growth of Bifidobacterium species and had no effect on potentially harmful gut organisms. Moreover, supplementation with a mixture of these six cyclic dipeptides significantly increased the abundance of microorganisms related to the genus Bifidobacterium in a human colonic microbiota model culture system, although supplementation with a single type of dipeptide had no effect. These results show that cyclic dipeptides containing Val-Leu and Val-Ile produced by C-3102 could serve as bifidogenic growth factors in the gut microbial community.

Effect of Bacillus subtilis C-3102 on bone mineral density in healthy postmenopausal Japanese women: a randomized, placebo-controlled, double-blind clinical trial.

Gut microbiota influence the host immune system and are associated with various diseases. In recent years, postmenopausal bone loss has been suggested to be related to gut microbiota. In the present study, we investigated the treatment effect of the probiotic Bacillus subtilis C-3102 (C-3102) on bone mineral density (BMD) and its influence on gut microbiota in healthy postmenopausal Japanese women. Seventy-six healthy postmenopausal Japanese women were treated with a placebo or C-3102 spore-containing tablets for 24 weeks. When compared with the placebo, C-3102 significantly increased total hip BMD (placebo = 0.83 ± 0.63%, C-3102 = 2.53 ± 0.52%, p=0.043). There was a significant group-by-time interaction effect for urinary type I collagen cross-linked N-telopeptide (uNTx) (p=0.033), a marker of bone resorption. Specifically, the C-3102 group showed significantly lower uNTx when compared with the placebo group at 12 weeks of treatment (p=0.015). In addition, in the C-3102 group, there was a trend towards a decrease in the bone resorption marker tartrate-resistant acid phosphatase isoform 5b (TRACP-5b) when compared with the placebo group at 12 weeks of treatment (p=0.052). The relative abundance of genus Bifidobacterium significantly increased at 12 weeks of treatment compared with the baseline in the C-3102 group. The relative abundance of genus Fusobacterium was significantly decreased in the C-3102 group at 12 and 24 weeks of treatment compared with the baseline. These data suggested that C-3102 improves BMD by inhibiting bone resorption and modulating gut microbiota in healthy postmenopausal women.

Maternal antimicrobial use at delivery has a stronger impact than mode of delivery on bifidobacterial colonization in infants: a pilot study.

OBJECTIVE: To investigate factors related to bifidobacterial colonization in early infancy, with a focus on maternal antimicrobial use at delivery. STUDY DESIGN: A cross-sectional pilot study was performed. Feces samples of 33 Japanese healthy infants were collected over 10 months and analyzed by next-generation sequencing to examine the diversity and abundance of the gut microbiota. RESULTS: The beta diversity index of the gut microbiota differed significantly based on maternal antimicrobial use at delivery (P < 0.05). The most dominant genus was bifidobacteria, and the relative abundance of bifidobacteria in infants exposed to maternal antibiotics was significantly lower than in those who were not exposed (P < 0.05). In contrast, the delivery mode showed no significant relationship with gut microbiota diversity. CONCLUSIONS: Maternal antimicrobial use at delivery has a stronger effect than delivery mode on the gut microbiota, especially for colonization of bifidobacteria.

Three extracellular dipeptidyl peptidases found in Aspergillus oryzae show varying substrate specificities.

Three extracellular dipeptidyl peptidase genes, dppB, dppE, and dppF, were unveiled by sequence analysis of the Aspergillus oryzae genome. We investigated their differential enzymatic profiles, in order to gain an understanding of the diversity of these genes. The three dipeptidyl peptidases were expressed using Aspergillus nidulans as the host. Each recombinant enzyme was purified and subsequently characterized. The enzymes displayed similar optimum pH values, but optimum temperatures, pH stabilities, and substrate specificities varied. DppB was identified as a Xaa-Prolyl dipeptidyl peptidase, while DppE scissile substrates were similar to the substrates for Aspergillus fumigatus DPPV (AfDPPV). DppF was found to be a novel enzyme that could digest both substrates for A. fumigatus DPPIV and AfDPPV. Semi-quantitative PCR revealed that the transcription of dppB in A. oryzae was induced by protein substrates and repressed by the addition of an inorganic nitrogen source, despite the presence of protein substrates. The transcription of dppE depended on its growth time, while the transcription of dppF was not affected by the type of the nitrogen source in the medium, and it started during the early stage of the fungal growth. Based on these results, we conclude that these enzymes may represent the nutrition acquisition enzymes. Additionally, DppF may be one of the sensor peptidases responsible for the detection of the protein substrates in A. oryzae environment. DppB may be involved in nitrogen assimilation control, since the transcription of dppB was repressed by NaNO3, despite the presence of protein substrates.

Diversity in mRNA expression of the serine-type carboxypeptidase ocpG in Aspergillus oryzae through intron retention.

Alternative splicing is thought to be a means for diversification of products by mRNA modification. Although some intron retentions are predicted by transcriptome analysis in Aspergillus oryzae, its physiological significance remains unknown. We found that intron retention occurred occasionally in the serine-type carboxypeptidase gene, ocpG. Analysis under various culture conditions revealed that extracellular nitrogen conditions influence splicing patterns; this suggested that there might be a correlation between splicing efficiency and the necessity of OcpG activity for obtaining a nitrogen source. Since further analysis showed that splicing occurred independently in each intron, we constructed ocpG intron-exchanging strain by interchanging the positions of intron-1 and intron-2. The splicing pattern indicated the probability that ocpG intron retention was affected by the secondary structures of intronic mRNA.

Serine-type carboxypeptidase KexA of Aspergillus oryzae has broader substrate specificity than Saccharomyces cerevisiae Kex1 and is required for normal hyphal growth and conidiation.

Aspergillus oryzae has an ortholog of Saccharomyces cerevisiae KEX1, termed kexA. A truncated form of KexA protein showed serine-type carboxypeptidase activity and somewhat broader substrate specificity than Kex1 protease. Furthermore, our results indicated that KexA is required for normal growth of A. oryzae and that it might be involved in hyphal branching.

Enzymatic properties of the recombinant serine-type carboxypeptidase OcpC, which is unique to Aspergillus oryzae.

Gene AO090103000153 is unique to Aspergillus oryzae RIB40 and A. flavus NRRL3357, and is speculated to encode a serine-type carboxypeptidase. In this study, we purified and characterized a heterologously expressed gene product of AO090103000153. 5'-Rapid amplification of cDNA ends indicated that the translation start site of the gene is located 1,586 bp downstream of the translation start site predicted by the genome sequencing project. The gene, starting from the revised translation start codon, termed ocpC, was transcribed constantly in A. oryzae RIB40. Purified recombinant OcpC exhibited the enzymatic properties of a serine-type carboxypeptidase. This protease was stable at temperatures below 45°C and a low pH, and had broad substrate specificity for N-acylpeptides, but it exhibited significantly lower specific activity and a lower k(cat) value for substrates than previously reported serine-type carboxypeptidases from A. oryzae.

Molecular cloning of ocpO encoding carboxypeptidase O of Aspergillus oryzae IAM2640.

Carboxypeptidase O from Aspergillus oryzae IAM2640 is a serine-type carboxypeptidase. In this study, we cloned and sequenced cDNA and genomic DNA carrying ocpO encoding carboxypeptidase O. The results showed that the length of ocpO was 1,816 bp, and the open reading frame encoded a putative preproenzyme composed of 472 amino acid residues of the mature carboxypeptidase O and an additional N-terminal sequence of 50 amino acid residues. A BLASTN search revealed that a gene, AO090020000351, in A. oryzae RIB40, which is strain used in genome-wide sequencing, is a homolog of ocpO. The difference between AO090020000351 and ocpO was only one nucleotide. The difference caused substitution of Ala for Pro at the 277th position of the enzyme; therefore the protein encoded by AO090020000351 was overproduced and purified. The purified protein showed enzymatic properties similar to carboxypeptidase O, indicating that carboxypeptidase O and protease encoded by AO090020000351 are same enzyme.

Heterologous expression and characterization of CpI, OcpA, and novel serine-type carboxypeptidase OcpB from Aspergillus oryzae.

In the genome of Aspergillus oryzae, 12 genes have been predicted to encode serine-type carboxypeptidases. However, the carboxypeptidase activities of the proteins encoded by these genes have not yet been confirmed experimentally. In this study, we have constructed three of these 12 genes overexpressing strains using Aspergillus nidulans and characterized their overproduced recombinant proteins. Of these three genes, one was previously named cpI; the other two have not been reported yet, and hence, we named them ocpA and ocpB. The recombinant proteins released amino acid residues from the C terminus of peptides, and the activity of the enzymes was inhibited by phenylmethylsulfonyl fluoride, indicating the enzymes to be serine-type carboxypeptidases. Recombinant OcpA, OcpB, and CpI were stable at 45 degrees C, 55 degrees C, and 55 degrees C, respectively, at a low pH. The enzymatic properties of recombinant OcpB were different from those of any reported serine-type carboxypeptidase. On the other hand, recombinant OcpA had similar enzymatic properties to A. oryzae carboxypeptidases O1 and O2. The DNA and N-terminal amino acid sequences of carboxypeptidases O1 and O2 from A. oryzae IAM2640 were similar to those of OcpA. Result of transcriptional analysis of ocpA, ocpB, and cpI suggest differences in transcriptional regulation between these genes.

Function analysis of steA homolog in Aspergillus oryzae.

The asexual ascomycete Aspergillus oryzae has an steA homolog in its genome. The steA homolog of A. oryzae was transcribed in the wild-type strain but steA disruption did not affect the phenotype of the disruptant strain. On the other hand, the steA-overexpressing strain showed the restriction of vegetative hyphal growth and conidiation, and the formation of balloon-shaped structures. The restriction of vegetative hyphal growth and conidiation were partially rescued by high osmolarity. In addition, the hyphal growth of the steA-overexpressing strain was inhibited by Calcofluor white, and the strain was found to be sensitive to cell-wall-degrading reagents. Increase of secretory cell-wall-degrading enzymes of the steA-overexpressing strain was detected. These results indicate that SteA regulates cell-wall-degrading enzymes and that the phenotypes of A. oryzae steA-overexpressing strain may be due to cell wall abnormalities. steA may participate in cell wall metabolism.