Abstract
Recently, fungal infections originating from the probiotic Saccharomyces ‘boulardii’ yeast are increasingly reported. Here, we aimed to reveal the background of and to diminish the virulence of this yeast, mitigating infection risks in vulnerable patient groups. Product and human isolates of S. ‘boulardii’ were subjected to in-host selection and their subclone lineages were compared phenotypically to identify target phenotypes and associated genes. More virulent isolates showed signs of selection for high osmotic tolerance in immunosuppressed mouse model, hence the genes NHA1 and ENA1 were deleted in six different ‘boulardii’ backgrounds. Only ENA1 deletion diminished virulence in our mouse fungemia model and it retained the ability for gut colonization and its probiotic characteristics, including similar effects on the gut microbiome in gavaged mice. We also demonstrated the successful substitution of the ENA1 gene with an antilisterial bacteriocin, opening a strategy for safe strains with therapeutic effect. Our strain development approach highlighted the importance of testing various genetic backgrounds and resulted in engineered strains with drastically reduced capability to cause bloodstream infections even in immunosuppressed hosts, establishing the groundwork for safer probiotic yeast therapies in the future.
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Data availability
Raw sequencing reads used in this study are deposited in NCBI SRA under BioProject PRJNA1165191 and PRJNA1358987. Cohort-called variant files are deposited in FigShare (https://doi.org/10.6084/m9.figshare.27105919). Raw data for each graph is included in Supplementary Data File. All additional data are available from the corresponding author on reasonable request.
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Acknowledgements
The research was funded by the National Research, Development and Innovation Office (NKFIH FK 138910 to W.P.P.); the Thematic Excellence Programme of the Ministry for Innovation and Technology in Hungary (TKP2021-EGA-20 to I.P.); the New National Excellence Program of the Ministry for Innovation (ÚNKP-18-3-I-DE-4, ÚNKP-19-3-I-DE-234, ÚNKP-20-3-II-DE-103, ÚNKP- 22-4-I-DE-293 to A.I.); the János Bolyai Research Scholarship of the Hungarian Academy of Sciences (BO/00227/20/8 to W.P.P. and BO/00294/25 to R.K.); and the National Institutes of Health (1DP2AT012795-01 to N.C.). We are grateful to Dr. Gábor Péter for providing the Listeria strain. Parts of this work were funded by the POC/2024/5/011 proof-of-concept grant of the University of Debrecen. Funding sources did not influence study design or interpretation and publication of the results. We thank two anonymous reviewers for their constructive suggestions throughout the review process.
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Conceptualization, W.P.P. and A.I.; methodology, A.I., W.P.P., Z.B., and N.C.; formal analysis, A.I., W.P.P., Á.J., R.K., F.N., L.F., D.B., N.B., and A.H.; resources, R.K., Z.B., L.M., I.P., and N.C.; data curation, A.I., W.P.P., and N.B.; writing—original draft preparation, A.I. and W.P.P.; writing—review and editing, A.I. and W.P.P.; visualization, A.I., W.P.P., and N.B.; supervision, W.P.P., I.P., and N.C.; project administration, W.P.P. and A.I.; funding acquisition, A.I., W.P.P., I.P., and N.C.
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The University of Debrecen and the North Carolina State University have submitted an international PCT patent application on December 10th, 2025 under application number PCT/2025/050102. The ENA1 deletion strain of PY0001 was deposited at (NCAIM) as Y 001536 as a strain for the purposes of patent procedure.
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Imre, A., Kovács, R., Jakab, Á. et al. ENA1 deficiency attenuates Saccharomyces ‘boulardii’ probiotic yeast virulence in immunosuppressed mouse fungaemia model. Commun Biol (2026). https://doi.org/10.1038/s42003-026-09763-z
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DOI: https://doi.org/10.1038/s42003-026-09763-z
