Expanding the therapeutic capacity of Saccharomyces cerevisiae var. boulardii for the treatment of gastrointestinal infections - ABSTRACT Gastrointestinal (GI) disease is a significant global problem, causing 3 million hospitalizations in the US alone. Conventional antimicrobial treatment of GI infections present challenges such as selection of antibiotic resistance and off-target interactions with commensal microbes. GI diseases are uniquely challenging to treat with protein- based therapies because they often cannot withstand the harsh and rapidly changing environment of the GI tract, which includes a rich collection of proteases. I hypothesize that an engineered probiotic, Saccharomyces cerevisiae var. boulardii (Sb), can deliver anti-infective, protein-based therapies to the GI tract to treat the key enteric pathogens Clostridioides difficile (Cd) and enterotoxigenic Bacteroides fragilis (ETBF). Next-generation therapies targeting Cd and ETBF, including antimicrobial peptides (AMPs), toxin-neutralizing binding domains, and anti-virulence proteins are in development and are amenable to Sb-based expression and secretion. Sb maintains viability throughout the mammalian GI tract and can be modified with sophisticated genetic engineering tools developed for the model yeast Saccharomyces cerevisiae. Herein, I propose to expand upon a genetic toolbox I have begun establishing to achieve finely tuned expression and secretion capacity from Sb in the con- text of the mammalian GI environment and use this toolbox to modulate the expression of a panel of anti-infective therapeutics targeted to either Cd or ETBF. I will thoroughly characterize a panel of Sb strains carrying these therapies in terms of efficacy, tolerability, and impact on the host microbiome in the context of enteric infection. The rationale for this proposal stems from i) the need for improved and controlled delivery strategies for anti- infective protein therapies to treat GI infections, and ii) the extensive potential for Sb, which has inherent anti- pathogen qualities, to be genetically modified for production and delivery of a large variety of therapeutic pay- loads. My central motivation is to demonstrate the capacity for engineered Sb to eradicate the clinically significant gut pathogens Cd and ETBF through therapeutic delivery systems that are both tunable and combinatorial. This will be achieved through two aims: 1) define and expand the therapeutic capacity of Sb specifically in the mam- malian gut environment by building a genetic toolbox of secretion constructs with varying secretion capacities of a panel of anti-Cd and anti-ETBF therapies with distinct mechanisms of action, and 2) demonstrate that engi- neered Sb is capable of selectively targeting the Cd and ETBF in murine models of infection. These experiments are significant as they generate novel treatment regimens for enteric infections, including against Cd which has significant morbidity and mortality worldwide, and for ETBF which has no clinically approved, targeted therapy available. This proposal is innovative as it will develop Sb as a carrier of anti-Cd and anti-ETBF AMPs for the first time and extensively define the impact of this therapeutic system on the host microbiota as well as the target pathogen. Finally, this proposal is impactful as it strives to develop a safe, non-invasive, and targeted treatment for major GI infections, and can be readily adapted to treat other maladies of the GI tract.
