Role of peptidyl-prolyl cis/trans-isomerases (PPlases) during uptake of binary bacterial toxins into the cytosol of mammalian cells

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Many bacterial toxins, such as diphtheria toxin, enter mammalian cells by receptor-mediated endocytosis and then deliver an enzymatic active moiety from endosomes into the cytosol, where it ADP-ribosylates specific cellular substrate molecules. This results in cell damage and the typical clinical symptoms of several important infectious diseases. The mechanisms of how the enzyme moieties translocate across endosomal membranes are not understood for most toxins and therefore of scientific and medical interest. We investigate how the enzyme components of binary actin-ADP-ribosylating toxins of Clostridium botulinum (C2 toxin), C. perfringens (Iota toxin) und C. difficile (CDT) translocate from endosomal vesicles into the cytosol. Here, a separate transport component forms pores in the membranes of acidified endosomes which serve as translocation channels for the respective unfolded enzyme components to reach the cytosol. We have discovered that besides the chaperone Hsp90, the activity of peptidyl-prolyl cis/trans isomerases (PPIases) is crucial for membrane translocation of the enzyme components. Moreover, we demonstrated for the first time that the enzyme component of C2 toxin (C2I), interacts with cyclophilins (CyPA, CyP-40) and FK506 binding proteins (FKBP 51/52) in vitro and in living cells. Cyclosporine A, an inhibitor of CyPs, as well as FK506, an inhibitor of FKBPs prevented translocation of the enzyme components from acidified endosomes into the cytosol and thereby protected cultured cells from intoxication by the binary actin-ADP-ribosylating toxins, but also from intoxication with diphtheria toxin. Prompted by these findings, we will investigate whether the discovered PPIase/Hsp90-dpendent translocation is specific for ADP-ribosylating toxins and focus on the mechanism by which PPIases and Hsp90 interact with ADP-ribosyltransferases. Most importantly, we will include the isolated recombinant ADP-ribosyltransferase domains of binary actin-ADP-ribosylating toxins in our ongoing studies in addition to their complete enzyme components. We will establish a novel approach to exploit the transport component of Anthrax toxins for delivery of the isolated ADP-ribosyltransferase domains into cells and to use specific inhibitors and antibodies to elucidate the role of CyPs, FKBPs and Hsp90 during membrane translocation of ADP-ribosyltransferases in more detail. Furthermore, we will investigate the role of CyPs and FKBPs during cellular uptake of diphtheria toxin. The suggested approaches will provide significant new knowledge on intracellular membrane transport of bacterial toxins. Prompted by these findings, we will screen novel non-immunosuppresive PPIase-inhibitors to develop and characterize substances that prevent uptake of bacterial ADP-ribosylating toxins into human cells. Such compounds could lead to alternative therapeutic strategies against toxin-producing (antibiotics-resistant) bacteria.