Abstract
Saccharomyces boulardii, a subspecies of Saccharomyces cerevisiae, is a well-known eukaryotic probiotic with many benefits for human health. In the present study, a recombinant strain of S. boulardii was prepared to use as a potential oral vaccine delivery vehicle. In this sense, a ura3 auxotroph strain of S. boulardii CNCM I-745 (known as S. cerevisiae HANSEN CBS 5926, Yomogi®) was generated using CRISPR/Cas9 methodology. Then a gene construct encoding a highly immunogenic protein, ovalbumin (OVA), was prepared and transformed into the ura3- S. boulardii. To facilitate the transport of the recombinant immunogen across the intestinal barrier, a claudin-targeting sequence from Clostridium perfringens enterotoxin (CPE) was added to the C-terminus of the expression cassette. The recombinant S. boulardii strain expressing the OVA-CPE fusion protein was then administered orally to a group of mice, and serum IgG and fecal IgA levels were evaluated by ELISA. Our results demonstrated that anti-OVA IgG in serum significantly increased in test group (P < 0.001) compared to control groups (receiving wild type S. boulardii or PBS), and the fecal IgA titer was significantly higher in test group (P < 0.05) than control groups. In parallel, a recombinant S. boulardii strain expressing the similar construct lacking C-terminal CPE was also administered orally. The result showed an increased level of serum IgG in group receiving yeasts expressing the CPE negative construct compared to control groups; however, the fecal IgA levels did not increase significantly. In conclusion, our findings indicated that the yeast S. boulardii, as a delivery vehicle with possible immunomodulatory effects, and c-CPE, as a targeting tag, synergistically assist to stimulate systemic and local immunity. This proposed recombinant S. boulardii system might be useful in the expression of other antigenic peptides, making it as a promising tool for oral delivery of vaccines or therapeutic proteins.
Highlights
MATERIALS AND METHODSThe yeast Saccharomyces boulardii is a known GRAS microorganism with the probiotic activity against a wide range of microbial pathogens in intestinal lumen (Czerucka et al, 2007)
S. boulardii resulted in several transformants on Yeast nitrogen base medium (YNB)-FOAU agar medium
To confirm the inactivation of ura3 gene, these transformants were grown on YNB and YNB-U media (Figure 4); the ura3 auxotrophs are not able to grow in YNB lacking uracil but grow normally in YNB-U
Summary
MATERIALS AND METHODSThe yeast Saccharomyces boulardii is a known GRAS (generally regarded as safe) microorganism with the probiotic activity against a wide range of microbial pathogens in intestinal lumen (Czerucka et al, 2007). Saccharomyces boulardii CNCM I-745 (Yomogi R ) was used for the construction of ura3 auxotroph mutant, the expression of OVA fusion protein, and animal studies. The episomal yeast plasmid, pYES2 (Invitrogen, United States), containing S. cerevisiae ura3 gene and the inducible Gal1 promoter was utilized as a control plasmid in transformation experiments as well as in preparation of expression cassettes.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
