Neutralizing C. difficile toxicity with probiotics that secrete rationally-designed inhibitory peptides.

ABSTRACT
Clostridioides difficile is a clinically important opportunistic pathogen that exploits disruptions in the commensal
microbiome of the gastrointestinal tract. C. difficile infection (CDI) is characterized by colitis and diarrhea, which
are largely caused by two secreted toxins, TcdA and TcdB. These primary virulence factors bind to the cell
surface via their CROPs and internalization domains; upon cell internalization - they glycosylate Rho-family
proteins, thus disrupting Rho-dependent cellular processes, ultimately leading to inflammation and increased
epithelial permeability. The threat of CDI is rising due to the prevalence of hypervirulent and antibiotic resistant
strains. The increasing risk of CDI, combined with the shortcomings of conventional antibiotic and fecal
transplantation treatment options, poses an urgent need for novel therapies. Biotherapeutic approaches using
monoclonal anti-TcdA/TcdB antibodies indicated a promising route, complementary to antibiotics; however, they
suffer from cumbersome administration and limitations in biodistribution.
Responding to these challenges, we developed neutralizing peptides that inhibit both TcdA and TcdB
and engineered probiotic yeast as a delivery vector of anti-toxin peptides in the colon. In our preliminary
work, we computationally designed and evaluated two experimentally effective neutralizing peptides (SA1 and
SB6), which demonstrated anti-TcdA and TcdB biorecognition and conferred epithelial protection in primary
derived human colonic epithelial monolayers. Additionally, we developed a yeast strain yielding >5 g/L peptide
during fermentation after knockout of key proteases.
In this work, we propose to implement this toolbox to develop a comprehensive strategy against
TcdB-mediated C. difficile infection. To this end, we will computationally design peptides to bind to several
domains on TcdB and evaluate a broader set of therapeutic hypotheses at the molecular, cellular, and organismal
scales. First, we will elucidate the molecular mechanism of TcdB inhibition by SB6. This knowledge will inform a
concerted computational-experimental design of peptides that target the TcdB active domain and neutralize its
Rho glycosylation activity. Next, we will use recently published crystal structures and known receptor-binding
domains of TcdB to develop peptides that block TcdB’s entry into the cell. These peptides will be inspired by the
proteins to which TcdB natively binds on the cell surface, as well as the reference CROP-blocking antibody
Bezlotoxumab. Finally, we will engineer probiotic yeast to secrete these peptides, and characterize their efficacy
on human colonic epithelial cell and murine animal C. difficile infection models. Collectively, these efforts will
elucidate the mechanisms by which inhibitory peptides can block TcdB activity and use this knowledge to develop
optimized peptides that inhibit CDI by integrating expertise at the atomic, molecular, cellular, and organismal
scales.