Revisiting an old antibiotic: bacitracin neutralizes binary bacterial toxins and protects cells from intoxication

Abstract

The antibiotic bacitracin (Bac) inhibits cell wall synthesis of gram‐positive bacteria. Here, we discovered a totally different activity of Bac: the neutralization of bacterial exotoxins. Bac prevented intoxication of mammalian cells with the binary enterotoxins Clostridium botulinum C2, C. perfringens ɩ, C. difficile transferase (CDT), and Bacillus anihracis lethal toxin. The transport (B) subunits of these toxins deliver their respective enzyme (A) subunits into cells. Following endocytosis, the B subunits form pores in membranes of endosomes, which mediate translocation of the A subunits into the cytosol. Bac inhibited formation of such B pores in lipid bilayers in vitro and in living cells, thereby preventing translocation of the A subunit into the cytosol. Bac preserved the epithelial integrity of toxin‐treated CaCo‐2 monolayers, a model for the human gut epithelium. In conclusion, Bac should be discussed as a therapeutic option against infections with medically relevant toxin‐producing bacteria, including C. difficile and B. anthracis, because it inhibits bacterial growth and neutralizes the secreted toxins.—Schnell, L., Felix, I., Müller, B., Sadi, M., von Bank, F., Papatheodorou, P., Popoff, M. R., Aktories, K., Waltenberger, E., Benz, R., Weichbrodt, C., Fauler, M., Frick, M., Barth, H. Revisiting an old antibiotic: bacitracin neutralizes binary bacterial toxins and protects cells from intoxication. FASEB J. 33, 5755–5771 (2019). http://www.fasebj.org

The Antibiotic Bacitracin Protects Human Intestinal Epithelial Cells and Stem Cell-Derived Intestinal Organoids fromClostridium difficileToxin TcdB

“…In a recent study, we already excluded that Bac inhibits endocytic mechanisms of cells [25]. In conclusion, Bac inhibits the pH-dependent membrane translocation of the catalytic subunit of TcdB, which is in accordance with the recent findings for other bacterial toxins that also deliver their catalytic subunit from acidic endosomes into the cytosol.…”

“…In conclusion, a set of experiments was performed to demonstrate the toxin-neutralizing effects of Bac towards C. difficile TcdB in the human intestinal epithelial cells and stem-cell derived human intestinal organoids from this important C. difficile toxin, which is a major contributor to CDAD. In combination with the recent observation that Bac neutralizes the actin-modifying toxin CDT from hypervirulent C. difficile strains [25], the findings might offer a starting point for the development of novel therapeutic options in the context of CDAD, in particular because Bac is already an approved drug in clinical use [24]. The implementation of iPSC-derived intestinal organoids could further fortify a potential role for Bac in the direct treatment of C. difficile -induced toxicity.…”

“…Transepithelial electrical resistance (TEER) measurements were performed by impedance spectrometry using the CellZScope (NanoAnalytics GmbH, Muenster, Germany) as described [25]. In brief, 3 × 10 4 CaCo-2 cells were seeded on 24-well cell culture inserts (Transwell® Permeable Supports, PET, 0.4 μ m from Corning GmbH, Wiesbaden, Germany) and incubated at 37°C and 5% CO 2 until a confluent monolayer was obtained.…”

“…Recently, we reported that Bac in addition to its antibacterial activity neutralizes a variety of medically relevant bacterial protein toxins and protects cultured human cells from intoxication by these toxins including binary toxins such as anthrax lethal toxin and the actin ADP-ribosylating toxin CDT of C. difficile [25]. We discovered that Bac prevents the pH-mediated transport of the enzyme subunits of these toxins across endosomal membranes into the cytosol of target cells, most likely by inhibiting the essential membrane transport function of their binding/transport subunits [25].…”

Research in a time of enteroids and organoids: how the human gut model has transformed the study of enteric bacterial pathogens

“…In addition to antibiotic properties, bacitracin was previously demonstrated to protect cultured human cells from intoxication by anthrax lethal toxin and CDT. 118 Zhu et al demonstrated that bacitracin protected Caco-2 and HIOs from TcdB activity, reducing downstream glucosylation of the target protein Rac1 and preventing depolymerization of F-actin in HIOs. 46 These experiments suggest that bacitracin, an approved drug for clinical use, has potential application to a combination therapy against C. difficile infection.…”