Human α-Defensins Inhibit Clostridium difficile Toxin B

Background & Aims: Clostridium difficile toxins A and B are major virulence factors implicated in pseudomembranous colitis and antibiotic-associated diarrhea. The toxins are glucosyltransferases, which inactivate Rho proteins involved in cellular signaling. Human α-defensins as part of the innate immune system inactivate various microbial pathogens as well as specific bacterial exotoxins. Here, we studied the effects of α-defensins human neutrophil protein (HNP)-1, HNP-3, and enteric human defensin (HD)-5 on the activity of C difficile toxins A and B. Methods: Inactivation of C difficile toxins by α-defensins in vivo was monitored by microscopy, determination of the transepithelial resistance of CaCo-2 cell monolayers, and analysis of the glucosylation of Rac1 in toxin-treated cells. In vitro glucosylation was used to determine Km and median inhibitory concentration (IC50) values. Formation of defensin-toxin complexes was analyzed by precipitation and turbidity studies. Results: Treatment of cells with human α-defensins caused loss of cytotoxicity of toxin B, but not of toxin A. Only α-defensins, but not β-defensin-1 or cathelicidin LL-37, inhibited toxin B–catalyzed in vitro glucosylation of Rho guanosine triphosphatases in a competitive manner, increasing Km values for uridine 5′-diphosphate-glucose up to 10-fold. The IC50 values for inhibition of toxin B–catalyzed glucosylation by the α-defensins were 0.6–1.5 μmol/L. At high concentrations, defensins (HNP-1 ≥2 μmol/L) caused high-molecular-mass aggregates, comparable to Bacillus anthracis protective antigen and lethal factor. Conclusion: Our data indicate that toxin B interacts with high affinity with α-defensins and suggest that defensins may provide a defense mechanism against some types of clostridial glucosylating cytotoxins.