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

许多细菌毒素，如白喉毒素，通过受体介导的内吞作用进入哺乳动物细胞，然后将酶活性部分从内体递送到胞质溶胶中，在胞质溶胶中，它使特定的细胞底物分子ADP-核糖基化。这导致细胞损伤和几种重要传染病的典型临床症状。对于大多数毒素，酶部分如何跨内体膜移位的机制尚不清楚，因此具有科学和医学意义。我们研究了肉毒梭菌（C2毒素）、C.产气荚膜杆菌（Iota毒素）和C.艰难梭菌（CDT）从内体囊泡易位到胞质溶胶中。在此，单独的转运组分在酸化的内体的膜中形成孔，所述孔充当相应未折叠的酶组分到达胞质溶胶的易位通道。我们已经发现，除了伴侣蛋白Hsp 90，肽基脯氨酰顺/反异构酶（PPIases）的活性是至关重要的酶组分的膜易位。此外，我们首次证明了C2毒素的酶组分（C2 I）与亲环素（CyPA，CyP-40）和FK 506结合蛋白（FKBP 51/52）在体外和活细胞中相互作用。环孢菌素A，CyPs的抑制剂，以及FK 506，FKBP的抑制剂，防止易位的酶组分从酸化的内体进入胞质溶胶，从而保护培养的细胞免受中毒的二元肌动蛋白-ADP-核糖基化毒素，但也从白喉毒素中毒。根据这些发现，我们将研究所发现的PPIase/Hsp 90依赖性易位是否对ADP-核糖基化毒素具有特异性，并重点研究PPIase和Hsp 90与ADP-核糖基转移酶相互作用的机制。最重要的是，我们将包括分离的重组ADP核糖基转移酶结构域的二元肌动蛋白ADP核糖基化毒素在我们正在进行的研究，除了他们的完整的酶成分。我们将建立一种新的方法来利用炭疽毒素的运输组件的ADP-核糖基转移酶域到细胞中的分离交付，并使用特定的抑制剂和抗体，以阐明在ADP-核糖基转移酶的膜转位的CyPs，FKBPs和Hsp 90的作用更详细。此外，我们将研究细胞摄取白喉毒素过程中CyPs和FKBPs的作用。这些方法将为细菌毒素的细胞内膜转运提供重要的新知识。根据这些发现，我们将筛选新型非免疫抑制性PPIase抑制剂，以开发和表征阻止细菌ADP核糖基化毒素摄入人体细胞的物质。这些化合物可能会导致针对产毒素（耐药性）细菌的替代治疗策略。

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.