Human alpha-defensins inhibit Clostridium difficile toxin B.
OBJECTIVE
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 alpha-defensins as part of the innate immune system inactivate various microbial pathogens as well as specific bacterial exotoxins. Here, we studied the effects of alpha-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 alpha-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 K(m) and median inhibitory concentration (IC(50)) values. Formation of defensin-toxin complexes was analyzed by precipitation and turbidity studies.


RESULTS
Treatment of cells with human alpha-defensins caused loss of cytotoxicity of toxin B, but not of toxin A. Only alpha-defensins, but not beta-defensin-1 or cathelicidin LL-37, inhibited toxin B-catalyzed in vitro glucosylation of Rho guanosine triphosphatases in a competitive manner, increasing K(m) values for uridine 5'-diphosphate-glucose up to 10-fold. The IC(50) values for inhibition of toxin B-catalyzed glucosylation by the alpha-defensins were 0.6-1.5 micromol/L. At high concentrations, defensins (HNP-1 > or = 2 micromol/L) caused high-molecular-mass aggregates, comparable to Bacillus anthracis protective antigen and lethal factor.


CONCLUSIONS
Our data indicate that toxin B interacts with high affinity with alpha-defensins and suggest that defensins may provide a defense mechanism against some types of clostridial glucosylating cytotoxins.
Giesemann
Torsten
T
Institut für Experimentelle and Klinische Pharmakologie und Toxikologie, Universität Freiburg, Freiburg, Germany.
Guttenberg
Gregor
G
Aktories
Klaus
K
D000818:Animals; D000890:Anti-Infective Agents / Q000378:metabolism* / Q000494:pharmacology; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D018938:Caco-2 Cells; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D017097:Electric Impedance; D004768:Enterotoxins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism* / Q000494:pharmacology; D006031:Glycosylation; D006801:Humans; D007413:Intestinal Mucosa / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D041701:Swiss 3T3 Cells; D013997:Time Factors; D023084:alpha-Defensins / Q000378:metabolism* / Q000494:pharmacology; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D000890:Anti-Infective Agents; D001426:Bacterial Proteins; D001427:Bacterial Toxins; C500225:DEFA5 protein, human; D004768:Enterotoxins; D004791:Enzyme Inhibitors; D023084:alpha-Defensins; C406413:human neutrophil peptide 1; C406427:human neutrophil peptide 3; C041348:tcdA protein, Clostridium difficile; C031815:toxB protein, Clostridium difficile; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; D020742:rhoA GTP-Binding Protein