Critical roles of a housekeeping sortase of probiotic Bifidobacterium bifidum in bacterium-host cell crosstalk.

Bifidobacterium bifidum YIT 10347 (BF-1) is adhesive in vitro. Here we studied the molecular aspects of the BF-1 adhesion process. We identified and characterized non-adhesive mutants and found that a class E housekeeping sortase was critical for the adhesion to mucin. These mutants were significantly less adhesive to GCIY cells than was the wild type (WT), which protected GCIY cells against acid treatment more than did a non-adhesive mutant. The non-adhesive mutants aberrantly accumulated precursors of putative sortase-dependent proteins (SDPs). Recombinant SDPs bound to mucin. Disruption of the housekeeping sortase influenced expression of SDPs and pilus components. Mutants defective in a pilin or in an SDP showed the same adhesion properties as WT. Therefore, multiple SDPs and pili seem to work cooperatively to achieve adhesion, and the housekeeping sortase is responsible for cell wall anchoring of its substrates to ensure their proper biological function.

Screening of Probiotic Candidates in Human Oral Bacteria for the Prevention of Dental Disease.

The oral cavity in healthy subjects has a well-balanced microbiota that consists of more than 700 species. However, a disturbance of this balance, with an increase of harmful microbes and a decrease of beneficial microbes, causes oral disorders such as periodontal disease or dental caries. Nowadays, probiotics are expected to confer oral health benefits by modulating the oral microbiota. This study screened new probiotic candidates with potential oral health benefits and no harmful effects on the oral cavity. We screened 14 lactobacillus strains and 36 streptococcus strains out of 896 oral isolates derived from healthy subjects. These bacteria did not produce volatile sulfur compounds or water-insoluble glucan, had higher antibacterial activity against periodontal bacteria, and had higher adherence activity to oral epithelial cells or salivary-coated hydroxyapatite in vitro. We then evaluated the risk of primary cariogenicity and infective endocarditis of the selected oral isolates. As a result, Lactobacillus crispatus YIT 12319, Lactobacillus fermentum YIT 12320, Lactobacillus gasseri YIT 12321, and Streptococcus mitis YIT 12322 were selected because they showed no cariogenic potential in an artificial mouth system and a lower risk of experimental infective endocarditis in a rat model. These candidates are expected as new probiotics with potential oral health benefits and no adverse effects on general health.

Protein synthesis in giant liposomes using the in vitro translation system of Thermococcus kodakaraensis.

An in vitro translation system, based on cell components of the hyperthermophilic archaeon, Thermococcus kodakaraensis, has previously been developed. The system has been optimized and applied for protein production at high temperatures (60-65 degrees C). In this paper, we have examined the possibilities to utilize this system at a lower temperature range using green fluorescence protein (GFP) as the reporter protein. By optimizing the composition of the reaction mixture, and adding chaperonins from the mesophilic Escherichia coli, the yield of protein production at 40 degrees C was increased by fivefold. For liposome encapsulation of the optimized system, water-in-oil cell-sized emulsions were prepared by adding the translation system/GFP mRNA mixture to mineral oil supplemented with 1,2-dioleoyl-sn -glycero-3-phosphatidylcholine (DOPC). Giant liposomes were formed when these emulsions passed across a water/oil interface occupied with DOPC. The liposomes were incubated at 40 degrees C for 90 min, and fluorescence was examined by laser confocal microscopy. A significant increase in average fluorescence intensity was observed in liposomes with GFP mRNA, but not in those without mRNA. Our results indicate that the T. kodakaraensis in vitro translation system is applicable for protein production within giant liposomes, and these artificial cell models should provide the methodology to reconstitute various cell functions from a constitutional biology approach.