An Engineered Synthetic Biologic Protects Against Clostridium difficile Infection

Abstract

Morbidity and mortality attributed to <i>Clostridium difficile</i> infection (CDI) have increased over the past 20 years. Currently, antibiotics are the only US FDA-approved treatment for primary <i>C. difficile</i> infection, and these are, ironically, associated with disease relapse and the threat of burgeoning drug resistance. We previously showed that non-toxin virulence factors play key roles in CDI, and that colonization factors are critical for disease. Specifically, a <i>C. difficile</i> adhesin, Surface Layer Protein A (SlpA) is a major contributor to host cell attachment. In this work, we engineered Syn-LAB 2.0 and Syn-LAB 2.1, two synthetic biologic agents derived from lactic acid bacteria, to stably and constitutively express a host-cell binding fragment of the <i>C. difficile</i> adhesin SlpA on their cell-surface. Both agents harbor conditional suicide plasmids expressing a codon-optimized chimera of the lactic acid bacterium's cell-wall anchoring surface-protein domain, fused to the conserved, highly adherent, host-cell-binding domain of <i>C. difficile</i> SlpA. Both agents also incorporate engineered biocontrol, obviating the need for any antibiotic selection. Syn-LAB 2.0 and Syn-LAB 2.1 possess positive biophysical and <i>in vivo</i> properties compared with their parental antecedents in that they robustly and constitutively display the SlpA chimera on their cell surface, potentiate human intestinal epithelial barrier function <i>in vitro</i>, are safe, tolerable and palatable to Golden Syrian hamsters and neonatal piglets at high daily doses, and are detectable in animal feces within 24 h of dosing, confirming robust colonization. In combination, the engineered strains also delay (in fixed doses) or prevent (when continuously administered) death of infected hamsters upon challenge with high doses of virulent <i>C. difficile</i>. Finally, fixed-dose Syn-LAB ameliorates diarrhea in a non-lethal model of neonatal piglet enteritis. Taken together, our findings suggest that the two synthetic biologics may be effectively employed as non-antibiotic interventions for CDI.