Brewing anti-toxin drugs using probiotic yeast

North Carolina State University Raleigh, Raleigh NC

Investigators

Abstract

Abstract Bacterial infections of the gut afflict millions of individuals worldwide. While treatment with antibiotics is currently highly effective, the increasing prevalence of antibiotic resistance is making these infections more difficult to treat. Furthermore, antibiotics can damage an individual’s health-promoting commensal bacteria, making them susceptible to C. difficile infections, which can be recurrent in 20% of cases. New drugs are therefore needed which can synergize with or prolong the utility of antibiotics. Bacteria commonly express toxins during infection, which play key roles in virulence by damaging host epithelial cells. In support of their importance, pathogen virulence is attenuated or eliminated entirely when their toxin genes are deleted. These toxins act through a variety of mechanisms, but one large and important family are the glycosyltransferase toxins, which cause cytopathic effects by attaching sugars (commonly glucose or ribose) to key locations on host proteins. A promising strategy, synergistic with antibiotics, is to neutralize the toxins, as this would halt the progression of disease and avoid off-target effects on commensal microbes. Unfortunately, toxin-neutralizing drugs do not exist for many bacterial pathogens. For the anti-toxin therapies that do exist, they can be prohibitively expensive, or target mutable regions of the toxins. In this work, we propose to develop peptides that neutralize the highly conserved enzymatic activity of bacterial toxins. To do so, we will exploit the observation that baker’s yeast (S. cerevisiae) is susceptible to these toxins. Because S. cerevisiae is so easy to engineer, it is therefore possible to screen massive peptide libraries and identify potent toxin inhibitors that rescue yeast growth. In fact, we have performed a pilot screen and have already identified a lead peptide inhibitor of C. difficile TcdB. We will first expand this screen to identify peptide inhibitors of 5 additional bacterial toxins. The potency of these inhibitors will be investigated in cell-based models of toxin activity, and the inhibitory mechanism of promising leads will be identified using in vitro assays, coupled with mass spectrometry. Finally, these leads will be encoded in the genome of probiotic yeast, enabling continuous biomanufacturing of these drugs at the site of disease. Probiotic yeast will also be engineered to display toxin binders on its cell surface, thereby sequestering additional toxin and preventing toxin contact with human cells. The efficacy of the peptides and yeast delivery vectors will be evaluated in animal models. Taken together, this work develops a generalizable platform for discovery, characterization, and delivery of anti-toxin therapeutics that has the potential to prolong the usability of existing antibacterial drugs.