CDT increases the adherence of in the gut by forming long protrusions in which the bacteria get caught. 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 2-Hydroxysaclofen 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. C2 toxin is a bacterial exotoxin and represents the prototype of the family of clostridial binary toxins which comprises amongst others the iota toxin and the CDT toxin (Barth and Aktories, 2011; Stiles, 2017). These toxins are secreted by the respective bacteria and consist of two non-linked proteins, the binding/translocation B-component, and the enzymatically active A-component. The B-component binds to a specific receptor on target cells and mediates the uptake of the A-component via receptor-mediated endocytosis. The B-component forms a pore into the endosomal membrane through which the A-component translocates into the cytosol. Here, the A-component covalently transfers an ADP-ribose moiety onto monomeric actin (G-actin), which leads to a depolymerization of the actin cytoskeleton and therefore to rounding of target cells (Reuner et al., 1987; Aktories and Wegner, 1992; Aktories et al., 2017b). All three toxins cause severe enterotoxic symptoms in humans or animals, which are the consequence of their enzymatic mode of action in cells. The C2 toxin causes necrosis and hemorrhagic lesions in the intestinal mucosa of mice (Simpson, 1982; Ohishi, 1983a,b) and fluid accumulation in the intestinal loop of pheasants and chicken (Kurazono et al., 1987). For the iota toxin, lambs and calves 2-Hydroxysaclofen have been identified as common casualties for its enterotoxicity (Songer, 1996; Billington et al., 1998). infections (CDI) are still on the rise in hospitals of Western countries and pose a severe threat due to life-threatening symptoms such as antibiotic-associated diarrhea or pseudomembranous colitis. CDT RAF1 has been identified as a novel virulence factor produced by hypervirulent strains and most likely contributes to an improved colonization of in the human gut (Aktories et al., 2018; Papatheodorou et al., 2018). The prototype of clostridial toxins, C2 toxin is composed of the A-component C2I and the B-component C2II (Ohishi, 1983a,b). After proteolytic activation of C2II, the resulting C2IIa forms ring-shaped heptamers that bind to carbohydrate structures, which have been found on the surface of all cell types, investigated so far (Barth 2-Hydroxysaclofen et al., 2000; Eckhardt et al., 2000). C2I attaches to specific motifs of the C2IIa heptamer and the C2IIa/C2I complex is taken up via receptor-mediated endocytosis (Barth et al., 1998a; Bl?cker et al., 2000; Kaiser et al., 2006). Acidification of the endosomal lumen results in formation of a C2IIa pore with a narrow inner diameter of 1C2 nm into the endosomal membrane (Barth et al., 2000; Schleberger et al., 2006). At least partial unfolding of C2I is required to translocate through the narrow C2IIa pore into the target cell cytosol where it ADP-ribosylates G-actin (Aktories et al., 1986; Haug et al., 2003b). We demonstrated earlier that translocation of C2I into the cytosol is facilitated.Every gray dot signal shown in Figure ?Figure11 represents an interaction event between C2I and Hsp90, Cyp40, or FKBP51, respectively. Hsp90 and Hsp70 and peptidyl-prolyl 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. C2 toxin is a bacterial exotoxin and represents the prototype of the family of clostridial binary toxins which comprises amongst others the iota toxin and the CDT toxin (Barth and Aktories, 2011; Stiles, 2017). These toxins are secreted by the respective bacteria and consist of two non-linked proteins, the binding/translocation B-component, and the enzymatically active A-component. The B-component binds to a specific receptor on target cells and mediates the uptake of the A-component via receptor-mediated endocytosis. The B-component forms a pore into the endosomal membrane through which the A-component translocates into the cytosol. Here, the A-component covalently transfers an ADP-ribose moiety onto monomeric actin (G-actin), which leads to a depolymerization of the actin cytoskeleton and therefore to rounding of target cells (Reuner et al., 1987; Aktories and Wegner, 1992; Aktories et al., 2017b). All three toxins cause severe enterotoxic symptoms in humans or animals, which are the consequence of their enzymatic mode of action in cells. The C2 toxin causes necrosis and hemorrhagic lesions in the intestinal mucosa of mice (Simpson, 1982; Ohishi, 1983a,b) and fluid accumulation in the intestinal loop of pheasants and chicken (Kurazono et al., 1987). For the iota toxin, lambs and calves have been identified as common casualties for its enterotoxicity (Songer, 1996; Billington et al., 1998). infections (CDI) are still on the rise in hospitals of Western countries and pose a severe threat due to life-threatening symptoms such as antibiotic-associated diarrhea or pseudomembranous colitis. CDT has been identified as a novel virulence factor produced by hypervirulent strains and most likely contributes to an improved colonization of in the human gut (Aktories et al., 2018; Papatheodorou et al., 2018). The prototype of clostridial toxins, C2 toxin is composed of the A-component C2I and the B-component C2II (Ohishi, 1983a,b). After proteolytic activation of C2II, the resulting C2IIa forms ring-shaped heptamers that bind to carbohydrate structures, which have been found on the surface of all cell types, investigated so far (Barth et al., 2000; Eckhardt et al., 2000). C2I attaches to specific motifs of the C2IIa heptamer and the C2IIa/C2I complex is taken up via receptor-mediated endocytosis (Barth et al., 1998a; Bl?cker et al., 2000; Kaiser et al., 2006). Acidification of the endosomal lumen results in formation of a C2IIa pore with a narrow inner diameter of 1C2 nm into the endosomal membrane (Barth et al., 2000; Schleberger et al., 2006). At least partial unfolding of C2I is required to translocate through the narrow C2IIa pore into the target cell cytosol where it ADP-ribosylates G-actin (Aktories et al., 1986; Haug et al., 2003b). We demonstrated earlier that translocation of C2I into the cytosol is facilitated not only by the C2IIa pore but requires activity of host cell chaperones and peptidyl-prolyl isomerases (PPIases) [for review see (Schiene-Fischer, 2015; Barth and Ernst, 2016; Ernst et al., 2017b; Schopf et al., 2017)]. We identified the heat shock protein Hsp90 and Hsp70 as well as isoforms of the cyclophilin (Cyp) and FK506 binding protein (FKBPs) family, namely CypA, Cyp40, and FKBP51, as specific interaction partners for C2I. Hsp90 and Hsp70 activities are ATP-dependent and play important roles during several cellular processes such as folding, refolding, avoiding aggregation of unfolded proteins as well as protein transport, e.g., through the endoplasmic reticulum or into mitochondria (Freeman and Morimoto, 1996; Chacinska et al., 2009;.