Dissecting the urokinase activation pathway using urokinase-activated anthrax toxin

Abstract

Anthrax toxin is a three-part toxin secreted by Bacillus anthracis, consisting of protective antigen (PrAg), edema factor (EF), and lethal factor (LF). To intoxicate host mammalian cells, PrAg, the cell-binding moiety of the toxin, binds to cells and is then proteolytically activated by furin on the cell surface, resulting in the active heptameric form of PrAg. This heptamer serves as a protein-conducting channel that translocates EF and LF, the two enzymatic moieties of the toxin, into the cytosol of the cells where they exert cytotoxic effects. The anthrax toxin delivery system has been well characterized. The amino-terminal PrAg-binding domain of LF (residues 1-254, LFn) is sufficient to allow translocation of fused "passenger" polypeptides, such as the ADP-ribosylation domain of Pseudomonas exotoxin A, to the cytosol of the cells in a PrAg-dependent process. The protease specificity of the anthrax toxin delivery system can also be reengineered by replacing the furin cleavage target sequence of PrAg with other protease substrate sequences. PrAg-U2 is such a PrAg variant, one that is selectively activated by urokinase plasminogen activator (uPA). The uPA-dependent proteolytic activation of PrAg-U2 on the cell surface is readily detected by western blotting analysis of cell lysates in vitro, or cell or animal death in vivo. Here, we describe the use of PrAg-U2 as a molecular reporter tool to test the controversial question of what components are required for uPAR-mediated cell surface pro-uPA activation. The results demonstrate that both uPAR and plasminogen play critical roles in pro-uPA activation both in vitro and in vivo.

Fig. 1

Binding and processing of pro-uPA and PrAg-U2 by HeLa and 293 cells. HeLa and 293 cells were cultured to confluence in 24-well plates, and preincubated with serum-free DMEM containing 2 mg/ml BSA, 1 μg/ml of Glu-plasminogen, with or without 10 μg/ml of PAI-1 for 30 min. Some cells were preincubated with serum-free DMEM containing 2 mg/ml BSA, 1 mM tranexamic acid, without plasminogen. Then 1 μg/ml each of pro-uPA and PrAg-U2 were added to the cells and incubated for the times indicated. The cells were thoroughly washed, and the cell lysates were analyzed by Western blotting using a monoclonal antibody against the uPA B-chain (#394) (upper panel), or by using a rabbit anti-PrAg polyclonal antibody (#5308) (lower panel) to determine the binding and processing status of pro-uPA and PrAg-U2.

Fig. 2

The cytotoxicity of PrAg-U2 to uPAR-expressing tumor cells is blocked by PAI-1. HeLa cells were cultured to 50% confluence, preincubated with serum-free DMEM containing 100 ng/ml of pro-uPA and 1 μg/ml of Glu-plasminogen with or without 2 μg/ml of PAI-1 for 30 min. Then PrAg and PrAg-U2 combined with FP59 (50 ng/ml) were added to the cells and incubated for 6 h. The toxins were removed and replaced with fresh serum-containing DMEM. MTT was added to determine cell viability at 48 h.

Fig. 3

uPA-dependent activation of PrAg-U2/FP59 requires the presence of uPA, uPAR, and plasminogen in vivo. (A) Plg, uPA, and uPAR-deficient mice are hyperresistant to uPA-activated anthrax toxin. Wild type mice and mice deficient in uPA, uPAR, and Plg were challenged with 200 μg PrAg-U2 with 10 μg FP59 intraperitoneally, and were monitored for disease. All wild type mice became terminally ill within 24 h of toxin administration, whereas no outwards or histological signs of toxicity were detected in uPA, uPAR, and Plg-deficient mice (P<0.01). (B) PAI-1-deficient mice are hypersensitive to PrAg-U2. PAI-1^−/− (open bars) or wild type control (solid bars) mice were challenged with varying concentrations of PrAg-U2 with 10 μg FP59, and monitored for disease. All PAI-1^−/− mice treated with 15 to 30 μg PrAg-U2 became terminally ill within 24 h of toxin administration, whereas no outwards or histological signs of toxicity were detected in wild type mice challenged with 30 μg PrAg-U2 (P<0.001). (C–J) Cell-surface uPA-dependent T-cell toxicity of PrAg-U2. Histological appearance of T cell regions of the spleen of wild type (C–G), uPA^−/− (H), uPAR^−/− (I), and Plg^−/− (J) mice 24 h after intraperitoneal injection of PBS (C) or 200 μg PrAg-U2 with 10 μg FP59 (D–J). Scattered clusters (examples indicated with arrows) of degenerating lymphocytes in wild type mice (D), absent in PBS-treated wild type mice (C), are identified as subpopulations of T-cells, by immunostaining with T-cell (E) and B-cell (F) antibodies, undergoing apoptosis as visualized by TUNEL-staining (G). (H–J) shows the absence of T-cell pathology in the spleens of uPA^−/− (H), uPAR^−/− (I) and Plg^−/− (J) mice. (C, D, and H–J) Hematoxylin/eosin staining. (Bars = 10 μm).