Chronic liver disease enables gut Enterococcus faecalis colonization to promote liver carcinogenesis.

Gut dysbiosis is observed in chronic hepatobiliary diseases and is frequently associated with liver carcinogenesis; however, the extent and specific mechanisms triggered by alterations in the microbiota mediating tumorigenesis in these patients remain unclear. Here we show that Enterococcus faecalis is abundant in the microbiota of patients with hepatitis C virus-related chronic liver disease. Xenotransplantation of gut microbiota from these patients increased the number of spontaneous liver tumors in mice and enhanced susceptibility to liver carcinogens. Hepatic colonization by gelE-positive E. faecalis increased liver expression of proliferative genes in a TLR4-Myd88-dependent manner, leading to liver tumorigenesis. Moreover, decreased fecal deoxycholic acid levels were associated with colonization by E. faecalis. Overall, these data identify E. faecalis as a key promoter of liver carcinogenesis.

Gut-derived Enterococcus faecium from ulcerative colitis patients promotes colitis in a genetically susceptible mouse host.

BACKGROUND: Recent metagenomic analyses have revealed dysbiosis of the gut microbiota of ulcerative colitis (UC) patients. However, the impacts of this dysbiosis are not fully understood, particularly at the strain level. RESULTS: We perform whole-genome shotgun sequencing of fecal DNA extracts from 13 healthy donors and 16 UC and 8 Crohn's disease (CD) patients. The microbiota of UC and CD patients is taxonomically and functionally divergent from that of healthy donors, with E. faecium being the most differentially abundant species between the two microbial communities. Transplantation of feces from UC or CD patients into Il10-/- mice promotes pathological inflammation and cytokine expression in the mouse colon, although distinct cytokine expression profiles are observed between UC and CD. Unlike isolates derived from healthy donors, E. faecium isolates from the feces of UC patients, along with E. faecium strain ATCC 19434, promotes colitis and colonic cytokine expression. Inflammatory E. faecium strains, including ATCC 19434 and a UC-derived strain, cluster separately from commercially available probiotic strains based on whole-genome shotgun sequencing analysis. The presence of E. faecium in fecal samples is associated with large disease extent and the need for multiple medications in UC patients. CONCLUSIONS: E. faecium strains derived from UC patients display an inflammatory genotype that causes colitis.

Multivalency effects of hemagglutinin component of type B botulinum neurotoxin complex on epithelial barrier disruption.

Hemagglutinin (HA) is one of the components of botulinum neurotoxin (BoNT) complexes and it promotes the absorption of BoNT through the intestinal epithelium by at least two specific mechanisms: cell surface attachment by carbohydrate binding, and epithelial barrier disruption by E-cadherin binding. It is known that HA forms a three-arm structure, in which each of three protomers has three carbohydrate-binding sites and one E-cadherin-binding site. A three-arm form of HA is considered to bind to these ligands simultaneously. In the present study, we investigated how the multivalency effect of HA influences its barrier-disrupting activity. We prepared type B full-length HA (three-arm form) and mini-HA, which is a deletion mutant lacking the trimer-forming domain. Size-exclusion chromatography analysis showed that mini-HA exists as dimers (two-arm form) and monomers (one-arm form), which are then separated. We examined the multivalency effect of HA on the barrier-disrupting activity, the E-cadherin-binding activity, and the attachment activity to the basolateral cell surface. Our results showed that HA initially attaches to the basal surface of Caco-2 cells by carbohydrate binding and then moves to the lateral cell surface, where the HA acts to disrupt the epithelial barrier. Our results showed that the multivalency effect of HA enhances the barrier-disrupting activity in Caco-2 cells. We found that basal cell surface attachment and binding ability to immobilized E-cadherin were enhanced by the multivalency effect of HA. These results suggest that at least these two factors induced by the multivalency effect of HA cause the enhancement of the barrier-disrupting activity.

Botulinum toxin A complex exploits intestinal M cells to enter the host and exert neurotoxicity.

To cause food-borne botulism, botulinum neurotoxin (BoNT) in the gastrointestinal lumen must traverse the intestinal epithelial barrier. However, the mechanism by which BoNT crosses the intestinal epithelial barrier remains unclear. BoNTs are produced along with one or more non-toxic components, with which they form progenitor toxin complexes (PTCs). Here we show that serotype A1 L-PTC, which has high oral toxicity and makes the predominant contribution to causing illness, breaches the intestinal epithelial barrier from microfold (M) cells via an interaction between haemagglutinin (HA), one of the non-toxic components, and glycoprotein 2 (GP2). HA strongly binds to GP2 expressed on M cells, which do not have thick mucus layers. Susceptibility to orally administered L-PTC is dramatically reduced in M-cell-depleted mice and GP2-deficient (Gp2(-/-)) mice. Our finding provides the basis for the development of novel antitoxin therapeutics and delivery systems for oral biologics.

Alliinase from Ensifer adhaerens and Its Use for Generation of Fungicidal Activity.

A bacterium Ensifer adhaerens FERM P-19486 with the ability of alliinase production was isolated from a soil sample. The enzyme was purified for characterization of its general properties and evaluation of its application in on-site production of allicin-dependent fungicidal activity. The bacterial alliinase was purified 300-fold from a cell-free extract, giving rise to a homogenous protein band on polyacrylamide gel electrophoresis. The bacterial alliinase (96 kDa) consisted of two identical subunits (48 kDa), and was most active at 60°C and at pH 8.0. The enzyme stoichiometrically converted (-)-alliin ((-)-S-allyl-L-cysteine sulfoxide) to form allicin, pyruvic acid, and ammonia more selectively than (+)-alliin, a naturally occurring substrate for plant alliinase ever known. The C-S lyase activity was also detected with this bacterial enzyme when S-alkyl-L-cysteine was used as a substrate, though such a lyase activity is absolutely absent in alliinase of plant origin. The enzyme generated a fungicidal activity against Saccharomyces cerevisiae in a time- and a dose-dependent fashion using alliin as a stable precursor. Alliinase of Ensifer adhaerens FERM P-19486 is the enzyme with a novel type of substrate specificity, and thus considered to be beneficial when used in combination with garlic enzyme with respect to absolute conversion of (±)-alliin to allicin.

Evaluation of uridine 5'-eicosylphosphate as a stimulant of cyclic AMP-dependent cellular function.

Sporulation of the yeast Saccharomyces cerevisiae is negatively regulated by cyclic AMP (cAMP). This microbial cell differentiation process was applied for the screening of a substance that can elevate the intracellular cAMP level. Among nucleoside 5'-alkylphosphates, uridine 5'-eicosylphosphate (UMPC20) selectively and predominantly inhibited ascospore formation of the yeast cells. We suppose the inhibitory effect of UMPC20 could indeed reflect the elevation of the cellular cAMP level.