Abstract
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.
Highlights
Clostridioides difficile (C. difficile), formerly known as Clostridium difficile, is a clinically highly relevant, Gram-positive anaerobic pathogen that releases several AB-type protein toxins
The large AB-type toxins TcdA and TcdB are considered the main virulence factors which cause the symptoms of C. difficile associated disease (CDAD) [1,2,3]
The molecular and cellular effects caused by CDT have been well characterized in recent years, its role as a virulence factor and, its contribution to disease are still under debate [1,9]. This is due to observations, for example, in a hamster model that C. difficile strains only expressing CDT but not TcdA or TcdB do not cause the typical symptoms of CDAD [12,13]
Summary
Clostridioides difficile (C. difficile), formerly known as Clostridium difficile, is a clinically highly relevant, Gram-positive anaerobic pathogen that releases several AB-type protein toxins. The molecular and cellular effects caused by CDT have been well characterized in recent years, its role as a virulence factor and, its contribution to disease are still under debate [1,9] This is due to observations, for example, in a hamster model that C. difficile strains only expressing CDT but not TcdA or TcdB do not cause the typical symptoms of CDAD [12,13]. CDTb alone caused cell rounding in Vero and CaCo-2 cells, as well as a decrease in the cell viability of Vero cells This cytotoxic effect depends on LSR, the specific cellular receptor of CDTb. Cell rounding was prevented by an enzymatically inactive CDTa mutant or by a cyclodextrin derivative, a known blocker of the transmembrane pores of other binary toxins [25]. Chloroquine inhibited cytotoxic effects of the combination of CDTa plus CDTb
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