The antimicrobial peptide Angie 5 inhibits TcdA and TcdB from Clostridioides difficile

Clostridioides (C.) difficile is a spore-forming, toxin-producing nosocomial human gut pathogen and a causative agent of gastrointestinal infections, leading to mild to severe diarrhea. Severe C. difficile infections (CDI) can cause life-threatening conditions, such as pseudomembranous colitis, colonic perforation, or toxic megacolon. The main virulence factors of C. difficile and responsible for CDI symptoms are two AB-type protein toxins, toxin A (TcdA) and toxin B (TcdB). TcdA and TcdB are large, single-chain proteins with multiple domains and glucosyltransferase activity. After receptor-mediated endocytosis, acidification of endosomes triggers insertion and pore formation of the toxins into the endosomal membrane for the delivery of their toxic glucosyltransferase domain (GTD) into the cytosol. There, the GTD glucosylates its target proteins, small GTPases of the Rho and/or Ras family, which leads amongst others to the collapse of the actin cytoskeleton and eventually to cell death. Here, we describe in silico predicted antimicrobial peptides, denoted as Angies, since they derive from the human endogenous protein angiogenin, as inhibitors for TcdA and TcdB. The strongest inhibitory capacity provided the derivative Angie 5, consistently in HeLa and Vero cells, as well as in the physiologically more relevant colon carcinoma cell line CaCo-2. Angie 5 delayed TcdA/TcdB-mediated glucosylation of its substrate proteins and, consequently, toxin-induced cell rounding as a consequence of actin-depolymerization. Moreover, the same Angie peptides that neutralized TcdA/TcdB also prevented the growth of C. difficile in vitro. In conclusion, our study paves the way for the development of antimicrobial peptide-based anti-toxin strategies to address C. difficile-associated diseases (CDADs).