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.

Functional role of surface layer proteins of Lactobacillus acidophilus L-92 in stress tolerance and binding to host cell proteins.

Lactobacillus acidophilus surface layer proteins (SLPs) self-assemble into a monolayer that is non-covalently bound to the outer surface of the cells. There they are in direct contact with the environment, environmental stressors and gut components of the host in which the organism resides. The role of L. acidophilus SLPs is not entirely understood, although SLPs seem to be essential for bacterial growth. We constructed three L. acidophilus L-92 strains, each expressing a mutant of the most abundant SLP, SlpA. Each carried a 12-amino acid c-myc epitope substitution at a different position in the protein. A strain was also obtained that expressed the SlpA paralog SlpB from an originally silent slpB gene. All four strains behaved differently with respect to growth under various stress conditions, such as the presence of salt, ox gall or ethanol, suggesting that SlpA affects stress tolerance in L. acidophilus L-92. Also, the four mutants showed differential in vitro binding ability to human host cell proteins such as uromodulin or dendritic cell (DC)-specific intercellular adhesion molecule-3 grabbing non-integrin (DC-SIGN). Furthermore, co-culture of murine immature DCs with a mutant strain expressing one of the recombinant SlpA proteins changed the concentrations of the cytokines IL-10 and IL-12. Our data suggest that SlpA and SlpB of L. acidophilus participate in bacterial stress tolerance and binding to uromodulin or DC-SIGN, possibly leading to effective immune-modification.

High levels of AID cause strand bias of mutations at A versus T in Burkitt's lymphoma cells.

Ig gene somatic hypermutation in the germinal center (GC) B cells occurs at C and G at roughly the same frequency. In contrast, there is a 2-fold increase of mutations at A relative to T on the non-transcribed strand of the V genes but it is unclear what triggers such strand bias. Using an efficient mutagenesis system that recapitulates characteristic features of Ig gene hypermutation in the GC B cells, we found that low levels of AID induced similar frequency of mutations at A and T. However, high levels of AID specifically increased mutations at A, but not T, leading to strand bias. These results explain why strand bias of A:T mutations is observed only in the highly mutated V genes but not in the less mutated switch region or the BCL-6 gene. High levels of AID also increased the proportion of transversions at G relative to transversions at C. Our results identify a clue to the strand bias of A:T mutations and provide an in vitro model to elucidate this unsolved mystery in the hypermutation field.

Analysis of mice deficient in both REV1 catalytic activity and POLH reveals an unexpected role for POLH in the generation of C to G and G to C transversions during Ig gene hypermutation.

Multiple DNA polymerases are involved in the generation of somatic mutations during Ig gene hypermutation. Mice expressing a catalytically inactive REV1 (REV1AA) exhibit reduction of both C to G and G to C transversions and moderate decrease of A/T mutations, whereas DNA polymerase η (POLH) deficiency causes greatly reduced A/T mutations. To investigate whether REV1 and POLH interact genetically and functionally during Ig gene hypermutation, we established REV1AA Polh(-/-) mice and analyzed Ig gene hypermutation in the germinal center (GC) B cells. REV1AA Polh(-/-) mice were born at the expected ratio and developed normally with no apparent gross abnormalities. B-cell development, maturation, Ig gene class switch and the GC B-cell expansion were not affected in these mice. REV1AA Polh(-/-) B cells also exhibited relatively normal sensitivity to etoposide and ionizing radiation. Analysis of somatic mutations in the J(H)4 intronic region revealed that REV1AA Polh(-/-) mice had a further decrease of overall mutation frequency compared with REV1AA or Polh(-/-) mice, indicating that the double deficiency additively affected the generation of mutations. Remarkably, REV1AA Polh(-/-) mice had nearly absent C to G and G to C transversions, suggesting that POLH is essential for the generation of residual C to G and G to C transversions observed in REV1AA mice. These results reveal genetic interactions between REV1 catalytic activity and POLH and identify an alternative pathway, mediated by non-catalytic REV1 and POLH, in the generation of C to G and G to C transversions.

Rapid cell division contributes to efficient induction of A/T mutations during Ig gene hypermutation.

Ig gene hypermutation is initiated by the activation-induced cytidine deaminase (AID), which converts cytosine to uracil and generates a U:G lesion. One of the unsolved mysteries is how AID-triggered U:G lesions result in efficient induction of mutations at non-damaged A/T bases in the V(H) genes of germinal center (GC) B cells. Genetic and biochemical evidence suggests that components of the mismatch repair pathway and the low fidelity DNA polymerase η are required for the induction of A/T mutations. However, mismatch repair proficient NIH3T3 cells are unable to generate a high frequency of A/T mutations, even after DNA polymerase η overexpression, suggesting that additional mechanisms are involved. Since GC B cells undergo enormous expansion while undergoing hypermutation, we hypothesized that rapid cell division might play a role in the induction of A/T mutations. To test this hypothesis, we utilized an efficient in vitro mutagenesis system, which closely mirrors physiological Ig gene hypermutation, in the human GC-like B cell line Ramos. Ramos cells transduced with AID-IRES-GFP retrovirus were cultured for 10 days in medium supplemented with 20% or 2% fetal bovine serum (FBS) to allow rapid and slow proliferation, respectively. Analysis of the V(H) gene mutations revealed that A/T mutations were significantly reduced in 2% FBS compared with 20% FBS, with transitions more affected than transversions. These results demonstrate that rapid cell division contributes to efficient induction of A/T mutations and suggest that the rate of DNA replication has a profound effect on the processing of AID-triggered U:G lesions.

Induction of a:T mutations is dependent on cellular environment but independent of mutation frequency and target gene location.

Based on its substrate specificity, activation-induced cytidine deaminase can directly induce C:G mutations in Ig genes. However the origin of A:T mutations, which occur in a similar proportion in germinal center (GC) B cells, is unclear. Genetic evidence suggests that the induction of A:T mutations requires the components of the mismatch repair system and DNA polymerase eta (POLH). We found that fibroblasts and GC B cells expressed similar levels of the mismatch repair components, but nonetheless the fibroblasts failed to generate a significant proportion of A:T mutations in a GFP reporter gene even after POLH overexpression. To investigate whether the ability to generate A:T mutations is dependent on the cellular environment (i.e., GC B cell or fibroblast) or the target gene (i.e., Ig or GFP), we developed a mutation detection system in a human GC-like cell line. We introduced a GFP gene with a premature stop codon into Ramos cells and compared the activation-induced cytidine deaminase-induced mutations in the endogenous V(H) and the transgenic GFP genes. Remarkably, a high proportion of A:T mutations was induced in both genes. Ectopic expression of POLH did not further increase the proportion of A:T mutations but diminished the strand bias of these mutations that is normally observed in V(H) genes. Intriguingly, the total mutation frequency in the GFP gene was consistently one-fifth of that in the V(H) gene. These results demonstrate that the ability to generate A:T mutations is dependent on the GC B cell environment but independent of the mutation frequency and target gene location.