Engineering probiotic yeasts to prevent and treat Clostridia-induced intestinal infections – TrojanYeast

Clostridioides difficile (Cd) and Clostridium perfringens (Cp) are often involved in severe intestinal infections. Both species secrete toxins that severely damage the intestinal epithelium. These infections are generally treated with antibiotics. However, resistant strains are emerging worldwide and alternative therapies are needed. Endolysins are highly promising weapons against pathogenic bacteria. They are the enzymes that bacteriophages use to degrade the bacterial cell wall and lyse the cell. Their direct or phage-mediated delivery at the point of infection is currently the main bottleneck. We lack of an appropriate vehicle.
Here, we propose to engineer the probiotic yeast S boulardii (Sb) to (i) secrete endolysins to kill the pathogens and (ii) antitoxin nanobodies to clean the environment by neutralizing the toxins released by the pathogens. We will construct collections of endolysins and of nanobodies, and screen them for pathogen killing efficiency, for toxin neutralizing efficacy, and for secretability in Sb. To construct, express and screen efficiently our libraries, we will use a modular cloning strategy for yeast, expand an existing bioreactor-based platform to quantify secreted protein levels, and develop an anaerobic co-culture platform for quantifying killing and neutralizing efficacities. Strains expressing selected endolysin/nanobody pairs will then be tested in mice models of disease for their prophylactic and therapeutic effects.
If successful, our project will pave the way for a novel type of treatment against Cd and Cp infections for human and animal health. This project will also lead to the development of a synthetic biology and technological platform for the rational engineering of probiotics against intestinal pathogens.