Characterization and Pharmacological Inhibition of the Pore-Forming Clostridioides difficile CDTb Toxin.

The clinically highly relevant Clostridioides (C.) difficile releases several AB-type toxins that cause diseases such as diarrhea and pseudomembranous colitis. In addition to the main virulence factors Rho/Ras-glycosylating toxins TcdA and TcdB, hypervirulent strains produce the binary AB-type toxin CDT. CDT consists of two separate proteins. The binding/translocation B-component CDTb facilitates uptake and translocation of the enzyme A-component CDTa to the cytosol of cells. Here, CDTa ADP-ribosylates G-actin, resulting in depolymerization of the actin cytoskeleton. We previously showed that CDTb exhibits cytotoxicity in the absence of CDTa, which is most likely due to pore formation in the cytoplasmic membrane. Here, we further investigated this cytotoxic effect and showed that CDTb impairs CaCo-2 cell viability and leads to redistribution of F-actin without affecting tubulin structures. CDTb was detected at the cytoplasmic membrane in addition to its endosomal localization if CDTb was applied alone. Chloroquine and several of its derivatives, which were previously identified as toxin pore blockers, inhibited intoxication of Vero, HCT116, and CaCo-2 cells by CDTb and CDTb pores in vitro. These results further strengthen pore formation by CDTb in the cytoplasmic membrane as the underlying cytotoxic mechanism and identify pharmacological pore blockers as potent inhibitors of cytotoxicity induced by CDTb and CDTa plus CDTb.

The cytotoxic effect of Clostridioides difficile pore-forming toxin CDTb.

Clostridioides (C.) difficile is clinically highly relevant and produces several AB-type protein toxins, which are the causative agents for C. difficile-associated diarrhea and pseudomembranous colitis. Treatment with antibiotics can lead to C. difficile overgrowth in the gut of patients due to the disturbed microbiota. C. difficile releases large Rho/Ras-GTPase glucosylating toxins TcdA and TcdB, which are considered as the major virulence factors for C. difficile-associated diseases. In addition to TcdA and TcdB, C. difficile strains isolated from severe cases of colitis produce a third toxin called CDT. CDT is a member of the family of clostridial binary actin ADP-ribosylating toxins and consists of two separate protein components. The B-component, CDTb, binds to the receptor and forms a complex with and facilitates transport and translocation of the enzymatically active A-component, CDTa, into the cytosol of target cells by forming trans-membrane pores through which CDTa translocates. In the cytosol, CDTa ADP-ribosylates G-actin causing depolymerization of the actin cytoskeleton and, eventually, cell death. In the present study, we report that CDTb exhibits a cytotoxic effect in the absence of CDTa. We show that CDTb causes cell rounding and impairs cell viability and the epithelial integrity of CaCo-2 monolayers in the absence of CDTa. CDTb-induced cell rounding depended on the presence of LSR, the specific cellular receptor of CDT. The isolated receptor-binding domain of CDTb was not sufficient to cause cell rounding. CDTb-induced cell rounding was inhibited by enzymatically inactive CDTa or a pore-blocker, implying that CDTb pores in cytoplasmic membranes contribute to cytotoxicity.

Human α-Defensin-5 Efficiently Neutralizes Clostridioides difficile Toxins TcdA, TcdB, and CDT.

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.

Human peptide α-defensin-1 interferes with Clostridioides difficile toxins TcdA, TcdB, and CDT.

The human pathogenic bacterium Clostridioides difficile produces two exotoxins TcdA and TcdB, which inactivate Rho GTPases thereby causing C. difficile-associated diseases (CDAD) including life-threatening pseudomembranous colitis. Hypervirulent strains produce additionally the binary actin ADP-ribosylating toxin CDT. These strains are hallmarked by more severe forms of CDAD and increased frequency and severity. Once in the cytosol, the toxins act as enzymes resulting in the typical clinical symptoms. Therefore, targeting and inactivation of the released toxins are of peculiar interest. Prompted by earlier findings that human α-defensin-1 neutralizes TcdB, we investigated the effects of the defensin on all three C. difficile toxins. Inhibition of TcdA, TcdB, and CDT was demonstrated by analyzing toxin-induced changes in cell morphology, substrate modification, and decrease in transepithelial electrical resistance. Application of α-defensin-1 protected cells and human intestinal organoids from the cytotoxic effects of TcdA, TcdB, CDT, and their combination which is attributed to a direct interaction between the toxins and α-defensin-1. In mice, the application of α-defensin-1 reduced the TcdA-induced damage of intestinal loops in vivo. In conclusion, human α-defensin-1 is a specific and potent inhibitor of the C. difficile toxins and a promising agent to develop novel therapeutic options against C. difficile infections.

Combined Pharmacological Inhibition of Cyclophilins, FK506-Binding Proteins, Hsp90, and Hsp70 Protects Cells From Clostridium botulinum C2 Toxin.

The Clostridium botulinum C2 toxin is an exotoxin causing severe enterotoxic symptoms. The C2 toxin consists of the binding/translocation component C2II, and the enzymatic active component C2I. After proteolytic activation, C2IIa forms heptamers that bind C2I. The C2IIa/C2I complex is taken up into mammalian target cells via receptor-mediated endocytosis. Acidification of endosomes leads to conformational changes in both components. C2IIa heptamers form a pore into the endosomal membrane, and C2I becomes unfolded and translocates through the narrow C2IIa pores into the cytosol of the cell. Here, C2I covalently transfers an ADP-ribose moiety from its co-substrate NAD+ onto G-actin, which leads to depolymerization of F-actin resulting in rounding up of adherent cells. Translocation of C2I into the cytosol depends on the activity of the chaperones Hsp90 and Hsp70 and peptidyl-prolyl cis/trans isomerases of the cyclophilin (Cyp) and FK506-binding protein (FKBP) families. Here, we demonstrated that C2I is detected in close proximity with Hsp90, Cyp40, and FKBP51 in cells, indicating their interaction. This interaction was dependent on the concentration of C2 toxin and detected in mammalian Vero and human HeLa cells. Moreover, the present study reveals that combination of radicicol, VER-155008, cyclosporine A, and FK506, which are specific pharmacological inhibitors of Hsp90, Hsp70, Cyps, and FKBPs, respectively, resulted in a stronger inhibition of intoxication of cells with C2 toxin compared to application of the single inhibitors. Thus, the combination of inhibitors showed enhanced protection of cells against the cytotoxic effects of C2 toxin. Cell viability was not significantly impaired by application of the inhibitor combination. Moreover, we confirmed that the combination of radicicol, VER-155008, CsA, and FK506 in particular inhibit the membrane translocation step of C2I into the cytosol whereas receptor binding and enzyme activity of the toxin were not affected. Our findings further characterize the mode of action of Hsp90, Hsp70, Cyps, and FKBPs during membrane translocation of bacterial toxins and furthermore supply starting points for developing of novel therapeutic strategies against diseases caused by bacterial toxins that depend on Hsp90, Hsp70, Cyps, and FKBPs.