Manipulating the growth environment through co-culture to enhance stress tolerance and viability of probiotic strains in the gastrointestinal tract.

IMPORTANCE: The viability of probiotics in the human gastrointestinal tract is important, as some reports indicate that the health benefits of live bacteria are greater than those of dead ones. Therefore, the higher the viability of the probiotic strain, the better it may be. However, probiotic strains lose their viability due to gastrointestinal stress such as gastric acid and bile. This study provides an example of the use of co-culture or pH-controlled monoculture, which uses more stringent conditions (lower pH) than normal monoculture to produce probiotic strains that are more resistant to gastrointestinal stress. In addition, co-cultured beverages showed higher viability of the probiotic strain in the human gastrointestinal tract than monocultured beverages in our human study.

Identification of genes essential for bile acid resistance in the probiotic Lacticaseibacillus paracasei strain Shirota.

Bile acid resistance is crucial to allow probiotic strains to survive in the gastrointestinal tract and exert health-promoting effects on their hosts. Our aim here was to determine the mechanism of this resistance via a genetic approach by identifying the genes essential for bile acid resistance in Lacticaseibacillus paracasei strain Shirota (LcS). We generated 4649 transposon-inserted lines of L. paracasei YIT 0291, which has the same genome sequence as LcS but lacks the pLY101 plasmid, and we screened them for bile-acid-sensitive mutants. The growth of 14 mutated strains was strongly inhibited by bile acid, and we identified 10 genes that could be involved in bile acid resistance. Expression of these genes was not markedly induced by bile acid, suggesting that their homeostatic expression is important for exerting bile acid resistance. Two mutants in which the transposon was independently inserted into cardiolipin synthase (cls) genes, showed strong growth inhibition. Disruption of the cls genes in LcS caused decreased cardiolipin (CL) production and the accumulation of the precursor phosphatidylglycerol in bacterial cells. These data suggest that LcS possesses several mechanisms for exerting bile acid resistance, and that homeostatic CL production is among the factors most essential for this resistance.

Diverse impact of a probiotic strain, Lacticaseibacillus paracasei Shirota, on peripheral mononuclear phagocytic cells in healthy Japanese office workers: a randomized, double-blind, controlled trial.

Mononuclear phagocytic cells (MPCs) are classified into monocytes (Mos)/macrophages and dendritic cells (DCs) based on their functions. Cells of MPCs lineage act as immune modulators by affecting effector cells, such as NK cells, T cells, and B cells. This study aimed to investigate the effects of Lacticaseibacillus paracasei strain Shirota (LcS) ingestion on peripheral MPCs, particularly on their expression of functional cell-surface molecules enhanced in healthy adults. Thus, twelve healthy office workers consumed a fermented milk drink containing 1.0 × 1011 cfu of LcS (LcS-FM) or a control unfermented milk drink (CM) once a day for 6 weeks. Peripheral blood mononuclear cells (PBMCs) were prepared from blood samples, and immune cells and functional cell-surface molecules were analyzed. We observed remarkable differences in the expression of HLAABC, MICA, CD40, and GPR43 in plasmacytoid DCs (pDCs) between the LcS-FM and CM groups, whereas no difference was found in CD86 or HLADR expression. The LcS-FM group exhibited higher CD40 expression in both conventional DCs (cDCs) and Mos, especially in type 2 conventional DCs (cDC2s) and classical monocytes (cMos); higher percentages of cMos, intermediate monocytes (iMos), and nonclassical monocytes; and higher numbers of cMos and iMos in PBMCs than the CM group. LcS ingestion increased the expression of HLAABC, MICA, CD40, and GPR43 in pDCs and CD40 in cDCs and Mos, particularly cDC2s and cMos. These results suggest that LcS modulates the function of MPCs that may lead to the regulation of immune effector functions in healthy adults.

Spatial distribution of live gut microbiota and bile acid metabolism in various parts of human large intestine.

Gut microbiomics is based on analysis of both live and dead cells in the stool. However, to understand the ecology of gut microbiota and their symbiotic relationships with hosts, spatial distribution of live bacteria must be examined. Here, we analyzed the live composition of luminal microbiota (LM) and mucosa-associated microbiota (MAM) in the ascending and descending colons and the rectums of 10 healthy adults and compared it with the total composition. The abundance of Lachnospiraceae in live LM decreased along the gut length and was significantly lower than that in total LM. Contrastingly, the abundance of Bacteroidaceae and Bifidobacteriaceae in live LM was higher than that in total LM, suggesting differences in death rate during gut migration. Live Enterobacteriaceae levels in MAM were significantly higher in rectum than in the ascending and descending colons and in LM. High-performance liquid chromatographic analysis of luminal bile acids revealed that 7α-dehydroxylation occurred towards the rectum. In live LM where a bile acid-inducible gene could be detected, 7α-dehydroxylation rates were higher than those in the group without the gene. Overall, we showed differences in live bacteria composition among three gut sites and between LM and MAM, highlighting the importance of understanding their spatial distribution.

Dynamic analysis of human small intestinal microbiota after an ingestion of fermented milk by small-intestinal fluid perfusion using an endoscopic retrograde bowel insertion technique.

Probiotic products have been shown to have beneficial effects on human hosts, but what happens in the gastrointestinal tract after its ingestion remains unclear. Our aim was to investigate the changes within the small intestines after a single intake of a fermented milk product containing a probiotic. We have periodically collected the small-intestinal fluids from the terminal ileum of seven healthy subjects for up to 7 h after ingestion by small-intestinal fluid perfusion using an endoscopic retrograde bowel insertion technique. The bacterial composition of the terminal ileum clearly revealed that the ingested probiotics (Lactobacillus casei strain Shirota: LcS and Bifidobacterium breve strain Yakult: BbrY) occupied the ileal microbiota for several hours, temporarily representing over 90% of the ileal microbiota in several subjects. Cultivation of ileal fluids showed that under a dramatic pH changes before reaching the terminal ileum, a certain number of the ingested bacteria survived (8.2 ± 6.4% of LcS, 7.8 ± 11.0% of BbrY). This means that more than 1 billion LcS and BbrY cells reached the terminal ileum with their colony-forming ability intact. These results indicate that there is adequate opportunity for the ingested probiotics to continuously stimulate the host cells in the small intestines. Our data suggest that probiotic fermented milk intake affects intestinal microbes and the host, explaining part of the process from the intake of probiotics to the exertion of their beneficial effects on the host.

Splicing of human chloride channel 1.

Expression of chloride channel 1 (CLCN1/ClC-1) in skeletal muscle is driven by alternative splicing, a process regulated in part by RNA-binding protein families MBNL and CELF. Aberrant splicing of CLCN1 produces many mRNAs, which were translated into inactive proteins, resulting in myotonia in myotonic dystrophy (DM), a genetic disorder caused by the expansion of a CTG or CCTG repeat. This increase in abnormal splicing variants containing exons 6B, 7A or the insertion of a TAG stop codon just before exon 7 leads to a decrease in expression of the normal splice pattern. The majority of studies examining splicing in CLCN1 have been performed using mouse Clcn1, as have investigations into the activation and suppression of normal splicing variant expression by MBNL1-3 and CELF3-6, respectively. In contrast, examinations of human CLCN1 have been less common due to the greater complexity of splicing patterns. Here, we constructed a minigene containing CLCN1 exons 5-7 and established a novel assay system to quantify the expression of the normal splicing variant of CLCN1 using real-time RT-PCR. Antisense oligonucleotides could promote normal CLCN1 alternative splicing but the effective sequence was different from that of Clcn1. This result differs from previous reports using Clcn1, highlighting the effect of differences in splicing patterns between mice and humans.

[Myotonic dystrophy].

No effective treatment was available for myotonic dystrophy, even in animal model. We have established a new antisense oligonucleotide delivery to skeletal muscle of mice with bubble liposomes, and led to increased expression of chloride channel (CLCN1) protein and the amelioration of myotonia. In other experiments, we also identified small molecule compounds that correct aberrant splicing of Clcn1 gene. Manumycin A corrected aberrant splicing of Clcn1 in mouse model.

Manumycin A corrects aberrant splicing of Clcn1 in myotonic dystrophy type 1 (DM1) mice.

Myotonic dystrophy type 1 (DM1) is the most common muscular dystrophy in adults and as yet no cure for DM1. Here, we report the potential of manumycin A for a novel DM1 therapeutic reagent. DM1 is caused by expansion of CTG repeat. Mutant transcripts containing expanded CUG repeats lead to aberrant regulation of alternative splicing. Myotonia (delayed muscle relaxation) is the most commonly observed symptom in DM1 patients and is caused by aberrant splicing of the skeletal muscle chloride channel (CLCN1) gene. Identification of small-molecule compounds that correct aberrant splicing in DM1 is attracting much attention as a way of improving understanding of the mechanism of DM1 pathology and improving treatment of DM1 patients. In this study, we generated a reporter screening system and searched for small-molecule compounds. We found that manumycin A corrects aberrant splicing of Clcn1 in cell and mouse models of DM1.