Cyclophilin-Facilitated Membrane Translocation as Pharmacological Target to Prevent Intoxication of Mammalian Cells by Binary Clostridial Actin ADP-Ribosylated Toxins

Abstract

Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin and Clostridium difficile CDT belong to the family of binary actin ADP-ribosylating toxins and are composed of a binding/translocation component and a separate enzyme component. The enzyme components ADP-ribosylate G-actin in the cytosol of target cells resulting in depolymerization of F-actin, cell rounding and cell death. The binding/translocation components bind to their cell receptors and form complexes with the respective enzyme components. After receptor-mediated endocytosis, the binding/translocation components form pores in membranes of acidified endosomes and the enzyme components translocate through these pores into the cytosol. This step is facilitated by the host cell chaperone heat shock protein 90 and peptidyl-prolyl cis/trans isomerases including cyclophilin A. Here, we demonstrate that a large isoform of cyclophilin A, the multi-domain enzyme cyclophilin 40 (Cyp40), binds to the enzyme components C2I, Ia and CDTa in vitro. Isothermal titration calorimetry revealed a direct binding to C2I with a calculated affinity of 101 nM and to Ia with an affinity of 1.01 μM. Closer investigation for the prototypic C2I revealed that binding to Cyp40 did not depend on its ADP-ribosyltransferase activity but was stronger for unfolded C2I. The interaction of C2I with Cyp40 was also demonstrated in lysates from C2-treated cells by pull-down. Treatment of cells with a non-immunosuppressive cyclosporine A derivative, which still binds to and inhibits the peptidyl-prolyl cis/trans isomerase activity of cyclophilins, protected cells from intoxication with C2, iota and CDT toxins, offering an attractive approach for development of novel therapeutic strategies against binary actin ADP-ribosylating toxins.

ADP-ribosylating toxins modifying the actin cytoskeleton

“…Interestingly, Hsp90 also coprecipitates with C2I from these lysates, and purified Hsp90 directly binds to C2I in vitro [112]. In contrast, the C2IIa component does not interact with Hsp90 and the PPIases in vitro [134].…”

“…The pretreatment of cells with PPIase-plus Hsp90-inhibitors more strongly protects cells from C2 toxins compared to an individual inhibitor, a hint that Hsp90 and the PPIases might synergistically facilitate C2I uptake into the cytosol [112]. By using specific antibodies, CypA, Cyp40, and FKBP51 were identified as directly interacting with C2I in vitro as demonstrated by dot blot assay, isothermal titration calorimetry, and coprecipitation with C2I from lysates of C2 toxin-treated cells [111,112,134]. Interestingly, Hsp90 also coprecipitates with C2I from these lysates, and purified Hsp90 directly binds to C2I in vitro [112].…”

“…CsA and FK506 (alias Tacrolimus) are licensed drugs for immunosuppression therapy [149][150][151][152][153]. However, targeted pharmacological inhibition of the PPIase activity of Cyps with the nonimmunosuppresive CsA-derivative VK112 [126,154] protects cells from intoxication with C2, iota and CDT toxins [134], as depicted in Figure 14.8. This substance, which acts on the molecular level of toxin uptake into cells, could be considered as a lead compound for developing novel therapeutic strategies against not only binary actin ADP-ribosylating toxins (e.g., food poisoning), but also during infections that include hypervirulent strains of C. difficile.…”

FKBPs in bacterial infections

“…Initially, a protective effect of CsA against intoxication binary toxins was observed (Dmochewitz et al, 2011). This, in the following, led to the identification human CypA and its multidomain homolog Cyp40 as interaction partners of these toxins which also facilitate their translocation from acidified endosomes into the host cell cytosol (Ernst et al, 2014;Kaiser et al, 2011). In a subsequent study, by using the toxic C2I subunit of botulinum toxin in co-precipitation experiments, FKBP51 was identified as another PPIase capable of binding binary toxins and promoting their translocation into the cytosol.…”

“…As the authors of the study suggest, it still remains to be solved whether host PPIases facilitate the translocation of bacterial binary toxins via their enzymatic activity, or rather their chaperone-like interaction. Nevertheless, the observed pharmacological effects of known PPIase inhibitors encourage the search for non-immunosuppresive drugs that can be applied in toxin-associated diseases (Ernst et al, 2014;Kaiser et al, 2012).…”