ENA1 deficiency attenuates Saccharomyces 'boulardii' probiotic yeast virulence in immunosuppressed mouse fungaemia model.

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.