Abstract
Infections with the pathogenic bacterium Clostridioides (C.) difficile are coming more into focus, in particular in hospitalized patients after antibiotic treatment. C. difficile produces the exotoxins TcdA and TcdB. Since some years, hypervirulent strains are described, which produce in addition the binary actin ADP-ribosylating toxin CDT. These strains are associated with more severe clinical presentations and increased morbidity and frequency. Once in the cytosol of their target cells, the catalytic domains of TcdA and TcdB glucosylate and thereby inactivate small Rho-GTPases whereas the enzyme subunit of CDT ADP-ribosylates G-actin. Thus, enzymatic activity of the toxins leads to destruction of the cytoskeleton and breakdown of the epidermal gut barrier integrity. This causes clinical symptoms ranging from mild diarrhea to life-threatening pseudomembranous colitis. Therefore, pharmacological inhibition of the secreted toxins is of peculiar medical interest. Here, we investigated the neutralizing effect of the human antimicrobial peptide α-defensin-5 toward TcdA, TcdB, and CDT in human cells. The toxin-neutralizing effects of α-defensin-5 toward TcdA, TcdB, and CDT as well as their medically relevant combination were demonstrated by analyzing toxins-induced changes in cell morphology, intracellular substrate modification, and decrease of trans-epithelial electrical resistance. For TcdA, the underlying mode of inhibition is most likely based on the formation of inactive toxin-defensin-aggregates whereas for CDT, the binding- and transport-component might be influenced. The application of α-defensin-5 delayed intoxication of cells in a time- and concentration-dependent manner. Due to its effect on the toxins, α-defensin-5 should be considered as a candidate to treat severe C. difficile–associated diseases.
Highlights
Bacterial AB-type protein toxins belong to the most toxic substances in nature and are able to cause a broad variety of severe diseases in humans and animals
The toxins serve as important virulence factors, which are directly linked to the clinical symptoms of human diseases as for example diphtheria, anthrax or other severe enteric complications such as medically relevant Clostridioides (C., formerly Clostridium) difficile– associated diseases, in particular diarrhea (CDAD)
Cells were maintained in their respective media (Vero cells: Minimum Essential Media (MEM) containing 10% fetal calf serum (FCS), 1 mM sodium pyruvate, 2 mM Lglutamine, 0.1 mM non-essential amino acids (NEAA), 10 g/L penicillin/streptomycin; Caco-2 cells: Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% FCS, 1 mM sodium pyruvate, 0.1 mM NEAA, 10 g/L penicillin/streptomycin) and split three times a week at a confluency of 80% to 100%
Summary
Bacterial AB-type protein toxins belong to the most toxic substances in nature and are able to cause a broad variety of severe diseases in humans and animals. The extraordinary potency of bacterial toxins is based on their inimitable structures harboring enzyme activities and their highly sophisticated uptake mechanisms The toxins serve as important virulence factors, which are directly linked to the clinical symptoms of human diseases as for example diphtheria, anthrax or other severe enteric complications such as medically relevant Clostridioides (C., formerly Clostridium) difficile– associated diseases, in particular diarrhea (CDAD). C. difficile infection (CDI) remains a remarkable challenge for affected patients and global health care systems. In addition to the patients suffering, CDIs remain a high economic burden. In England, the costs for CDIs have been estimated at €5000 - €15000 per case (Kuijper et al, 2006) accompanied with an increased length of stay in hospital (van Kleef et al, 2014; Wilcox et al, 2017)
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