Evaluation of the delivery of an anti-Listeria endolysin via CRISPR-Cas9 engineered probiotic Saccharomyces boulardii

<jats:sec> <jats:title>Abstract</jats:title> <jats:p> Listeriosis is a foodborne infection caused by <jats:italic>Listeria monocytogene</jats:italic> s that causes febrile gastroenteritis and central nervous system infections and that can often lead to fatality. Upon consumption of contaminated food, <jats:italic>Listeria</jats:italic> is able to survive a number of gastrointestinal stressors, including competition with the host microbiota. The emergence of antibiotic-resistant clones of <jats:italic>L. monocytogene</jats:italic> s, together with the side effects of antibiotic treatment, highlights the need for alternatives or additives for its treatment and prevention. <jats:italic>Saccharomyces boulardii</jats:italic> is a probiotic yeast that is often used alongside antibiotics to minimize side effects since it is not affected by them as a result of its eukaryotic nature. Furthermore, it can be engineered to produce a wide range of molecules. We previously engineered <jats:italic>Saccharomyces cerevisiae</jats:italic> through CRISPR-Cas9 integration to produce Ply511, a bacteriophage endolysin active against <jats:italic>L. monocytogene</jats:italic> s, showing the potential of engineered yeast to produce endolysins for biocontrol. In this study, we extended this approach to the probiotic yeast <jats:italic>S. boulardii</jats:italic> and directly compared the two yeasts as secretion hosts for Ply511. Using a simulated human gastrointestinal environment, we evaluated their ability to retain endolysin activity and reduce <jats:italic>L. monocytogenes</jats:italic> levels. We then tested the cell extracts from both yeasts in a bacterial consortium termed SImplified HUman intestinal MIcrobiota (SIHUMI), confirming a specificity for <jats:italic>Listeria</jats:italic> . Finally, we evaluated their activity in a simulated intestinal fermentation using fecal samples from human donors. Overall, this study demonstrates the potential of delivering endolysins to the gut via engineered probiotic <jats:italic>S. boulardii.</jats:italic> </jats:p> </jats:sec> <jats:sec> <jats:title>Key points</jats:title> <jats:p> <jats:list list-type="bullet"> <jats:list-item> <jats:p> <jats:italic>CRISPR-Cas9-engineered S. boulardii and S. cerevisiae were compared, both allowing the expression and activity of endolysin Ply511 against L. monocytogenes.</jats:italic> </jats:p> </jats:list-item> <jats:list-item> <jats:p> <jats:italic>Endolysin Ply511 retained its activity against L. monocytogenes in simulated gastrointestinal digestion and was specific against Listeria in a bacterial consortium termed SImplified HUman intestinal MIcrobiota (SIHUMI).</jats:italic> </jats:p> </jats:list-item> <jats:list-item> <jats:p> <jats:italic>Using fecal samples from human donors, the anti-Listeria effect was reduced potentially due to the lower metabolic activity of S. boulardii and the higher competition with the intestinal microbiome.</jats:italic> </jats:p> </jats:list-item> </jats:list> </jats:p> </jats:sec> <jats:sec> <jats:title>Graphical Abstract</jats:title> </jats:sec>