Tissue injury that accompanies hypoxemia/reoxygenation shares features with the host response in inflammation, suggesting that cytokines, such as IL-1, may act as mediators in this setting. Human endothelial cells (ECs) subjected to hypoxia (Po2 12-14 Torr) elaborated IL-1 activity into conditioned media in a time-dependent manner; this activity was completely neutralized by an antibody to IL-la. Production of IL-1 activity by hypoxic ECs was associated with an increase in the level of mRNA for IL-ia, and was followed by induction of endothelial-leukocyte adhesion molecule-1 and enhanced expression of intercellular adhesion molecule-1 (ICAM-1 ) during reoxygenation. During reoxygenation there was a three-to fivefold increased adherence of leukocytes, partly blocked by antibodies to endothelial-leukocyte adhesion molecule-1 and ICAM-1. Suppressing endothelial-derived IL-1, using either antibodies to IL-la, specific antisense oligonucleotides or the IL-1 receptor antagonist, decreased leukocyte adherence to reoxygenated ECs, emphasizing the integral role of IL-1 in the adherence phenomenon. Mice subjected to hypoxia (Po2 30-40 Torr) displayed increased plasma levels of IL-la, induction of IL-ia mRNA in the lung, and enhanced expression of ICAM-1 in pulmonary tissue compared with normoxic controls. These data suggest that hypoxia is a stimulus which induces EC synthesis and release of IL-la, resulting in an autocrine enhancement in the expression of adhesion molecules. (J.
SummaryThe binary Clostridium botulinum C2 toxin consists of the binding/translocation component C2IIa and the separate enzyme component C2I, which mono-ADP-ribosylates actin in eukaryotic cells. Pore formation of C2IIa in early endosomal membranes facilitates translocation of unfolded C2I into the cytosol. We discovered earlier that translocation of C2I depends on the activity of the host cell chaperone heat shock protein Hsp90. Here, we demonstrate that cyclosporin A, which inhibits the peptidyl-prolyl cis/trans isomerase activity of cyclophilins, inhibited intoxication of cells with C2 toxin and prevented uptake of C2I into the cytosol. Cyclosporin A blocked the pH-dependent translocation of C2I activity across membranes of intact cells and of partially purified early endosomes. In vitro, the addition of cytosol to C2 toxin-loaded endosomes induced translocation of C2I activity into the cytosol, which was prevented by pretreatment of the cytosol with an antibody against cyclophilin A. Pull-down experiments with lysates from C2 toxin-treated cells revealed specific binding of cyclophilin A to the N-terminal domain of C2I. In conclusion, our results suggest an essential role of cyclophilin A for translocation of C2I across endosomal membranes during the uptake of C2 toxin into mammalian cells.
SummaryThe C3 transferases from Clostridium botulinum (C3bot) and Clostridium limosum (C3lim) mono-ADP-ribosylate and thereby inactivate RhoA, -B and -C of eukaryotic cells. Due to their extremely poor cellular uptake, C3 transferases were supposed to be exoenzymes rather than exotoxins, challenging their role in pathogenesis. Here, we report for the first time that low concentrations of both C3lim and C3bot are selectively internalized into macrophages/monocytes in less than 3 h, inducing the reorganization of the actin cytoskeleton by ADP-ribosylation of Rho. We demonstrate that C3 transferases are internalized into the cytosol of macrophages/monocytes via acidified early endosomes. Bafilomycin A1, an inhibitor of endosomal acidification, protected J774A.1 macrophages and human promyelotic leukaemia cells (HL-60) from intoxication by C3. Moreover, confocal laser scanning microscopy revealed colocalization of C3 with early endosomes. An extracellular acidic pulse enabled direct translocation of cell surface-bound C3 across the cytoplasmic membrane to the cytosol. In line with this finding, both C3 proteins exhibited membrane activity in lipid bilayer membranes only under acidic conditions (pH < 5.5). In conclusion, we identified macrophages/monocytes as target cells for clostridial C3 transferases and shed light on their selective uptake mechanism, which might contribute to understand the role of C3 transferases in pathogenesis.
Some hypervirulent strains of Clostridium difficile produce the binary actin-ADP-ribosylating toxin C. difficile transferase (CDT) in addition to Rho-glucosylating toxins A and B. It has been suggested that the presence of CDT increases the severity of C. difficile-associated diseases, including pseudomembranous colitis. CDT contains a binding and translocation component, CDTb, that mediates the transport of the separate enzyme component CDTa into the cytosol of target cells, where CDTa modifies actin. Here we investigated the mechanism of cellular CDT uptake and found that bafilomycin A1 protects cultured epithelial cells from intoxication with CDT, implying that CDTa is translocated from acidified endosomal vesicles into the cytosol. Consistently, CDTa is translocated across the cytoplasmic membranes into the cytosol when cell-bound CDT is exposed to acidic medium. Radicicol and cyclosporine A, inhibitors of the heat shock protein Hsp90 and cyclophilins, respectively, protected cells from intoxication with CDT but not from intoxication with toxins A and B. Moreover, both inhibitors blocked the pH-dependent membrane translocation of CDTa, strongly suggesting that Hsp90 and cyclophilin are crucial for this process. In contrast, the inhibitors did not interfere with the ADP-ribosyltransferase activity, receptor binding, or endocytosis of the toxin. We obtained comparable results with the closely related iota-toxin from Clostridium perfringens. Moreover, CDTa and Ia, the enzyme component of iota-toxin, specifically bound to immobilized Hsp90 and cyclophilin A in vitro. In combination with our recently obtained data on the C2 toxin from C. botulinum, these results imply a common Hsp90/cyclophilin A-dependent translocation mechanism for the family of binary actin-ADP-ribosylating toxins.Clostridium difficile infection causes human diseases ranging from mild diarrhea to severe and potentially life-threatening pseudomembranous colitis. C. difficile-associated diseases occur in patients treated with broad-spectrum antibiotics. Under these conditions, the disturbed gut flora allows germination of C. difficile spores and colonization of the gut by this pathogen. C. difficile produces two exotoxins, toxin A (308 kDa) and toxin B (270 kDa), which are the causative agents of pseudomembranous colitis. The toxin-catalyzed glucosylation of Rho, Rac, and Cdc42 results in the inhibition of GTPase-mediated cell signaling, destruction of the actin cytoskeleton, and cell rounding that are the reasons for loss of integrity of the intestinal wall (for a review, see references 20 and 21).During the last 10 years, hypervirulent C. difficile strains were identified that produce, in addition to toxins A and B, a third exotoxin, the binary C. difficile transferase (CDT). Up to 35% of the strains tested produce CDT, and it has been suggested that the presence of CDT correlates with the severity of C. difficile-associated diseases (12,14,25,27,52). CDT belongs to the family of binary actin-ADP-ribosylating toxins and consists of two nonlink...
Summary The binary Clostridium botulinum C2 toxin consists of the binding/translocation component C2IIa and the separate enzyme component C2I. C2IIa delivers C2I into the cytosol of eukaryotic target cells where C2I ADP‐ribosylates actin. After receptor‐mediated endocytosis of the C2IIa/C2I complex, C2IIa forms pores in membranes of acidified early endosomes and unfolded C2I translocates through the pores into the cytosol. Membrane translocation of C2I is facilitated by the activities of host cell chaperone Hsp90 and the peptidyl‐prolyl cis/trans isomerase (PPIase) cyclophilin A. Here, we demonstrated that Hsp90 co‐precipitates with C2I from lysates of C2 toxin‐treated cells and identified the FK506‐binding protein (FKBP) 51 as a novel interaction partner of C2I in vitro and in intact mammalian cells. Prompted by this finding, we used the specific pharmacological inhibitor FK506 to investigate whether the PPIase activity of FKBPs plays a role during membrane translocation of C2 toxin. Treatment of cells with FK506 protected cultured cells from intoxication with C2 toxin. Moreover, FK506 inhibited the pH‐dependent translocation of C2I across membranes into the cytosol but did not interfere with the enzyme activity of C2I or binding of C2 toxin to cells. Furthermore, FK506 treatment delayed intoxication with the related binary actin ADP‐ribosylating toxins from Clostridium perfringens (iota toxin) and Clostridium difficile (CDT) but not with the Rho‐glucosylating Clostridium difficile toxin A (TcdA). In conclusion, our results support the hypothesis that clostridial binary actin‐ADP‐ribosylating toxins share a specific FKBP‐dependent translocation mechanism during their uptake into mammalian cells.
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