Fig 1.
CMG2 receptor-dependent in vivo toxicity of anthrax edema toxin.
A. CMG2-KO mice and their littermate WT control mice were challenged with 20 µg ET (I.P.), and monitored for signs of malaise and survival for 200 hours. Log-rank test for survival comparison. B. ET-induced cAMP increases in mice. Eighteen hours after challenge with 30 µg ET (I.V.), the mice (CMG2-KO and the controls) were euthanized, and blood, liver, heart, kidneys were collected and processed for measuring cAMP levels. Unpaired two-tailed Student’s t-test.
Fig 2.
PKA-independent in vivo toxicity of anthrax edema toxin.
A. cAMP-increasing bacterial toxins and cAMP-activated pathways. B. Activation of PKA by ET and forskolin. NIH3T3 cells were treated with ET (1 μg/mL) or FSK (forskolin, 10 μM) for various lengths of time as indicated with/without the presence of H89 (0.1 mM). The cell lysates were then analyzed by Western blotting using anti-phospho-CREB and anti-tubulin antibodies. Reprehensive of two independent experiments with similar results. C. Systemic administration of H89 effectively prevents ET-induced activation of CREB in the hearts of the ET-challenged mice. Mice were challenged with 30 µg ET (I.V.) or 30 µg ET (I.V.) plus 500 µg H89 (I.P.). Six 6 hours after administration, mice were euthanized and the heart tissue lysates prepared for Western blotting analysis using the antibodies as indicated (50 µg proteins loaded per lane). Of note, H89 could effectively diminish the ET-induced CREB activation. D. PKA inhibitor could not prevent ET-induced mortality. Mice were administered 30 µg ET (I.V.) or 500 µg H89 (I.P.) plus 30 µg ET (I.V.) and monitored for survival as indicated. E. CFTR-null (CFTR-KO (FABP-CFTR+)) mice and their WT control mice were challenged with 30 µg ET (I.V.) and monitored for survival. F. CFTR inhibitor could not prevent the ET-induced mortality. The mice were administered 30 µg ET (I.V.). 24 h and 1 h prior to ET injection, the mice were injected with PBS or 50 μg CFTRinh-172. (D-F) Log-rank test for survival comparison.
Fig 3.
The extracellular accumulation of cAMP is dispensable for the ET-induced cell rounding morphological change.
A, B. Toxin receptor-dependent cell rounding effect of ET. PR230 (a PA receptor-deficient CHO mutant cell line) and PR230 (CMG2) (PR230 reconstituted with CMG2 receptor) were incubated with/without ET (0.1 or 1.0 µg/mL) as indicated for 2 h. The ET-induced spherical morphological change was observed on PR230 (CMG2) cells but not on PR230 cells (A). Similarly, the ET-induced extracellular cAMP accumulation was only detected in the conditioned medium of PR230 (CMG2) cells but not that of PR230 cells (B). Means ± SD of three independent biological replicates. C. Co-culture experiment indicates no cell rounding effect of extracellular cAMP. The cover slips grown with PR230 cells and PR230 (CMG2) cells were placed in the same wells of a 6-well plate and co-incubated with/without ET for 2 h. Of note, ET could only induce the characteristic cell rounding morphological change to PR230 (CMG2) cells but not to PR230 cells in the co-culture system.
Fig 4.
ET-induced cAMP production, ATP depletion, and cytotoxicity to NIH3T3 cells.
A-C. The cells were incubated with/without ET for various lengths of time, followed by the measurements of cellular (A) and extracellular (B) cAMP levels, along with the cellular ATP levels (C). Means ± SD of three independent biological replicates. D. Cytotoxicity of ET and LT to NIH3T3 cells. Cells were incubated with various concentrations of PA in the presence of EF (1 μg/mL) or LF (1 μg/mL) for 48 h, followed by an MTT assay to assess cell viability. Means ± SD of three independent biological replicates.
Fig 5.
ET-induced cytotoxicity and cellular ATP depletion to multiple cell lines.
A-D. Cytotoxicity of ET and LT to multiple cell lines. MDA-MB-231 (A), B16-F10 (B), Glioma 261 (C) cells, and mouse primary fibroblasts (D) were incubated with various concentrations of PA in the presence of EF (1 μg/mL) or LF (1 μg/mL) for 48 h, followed by an MTT assay to assess the cell viability. E. Cytotoxicity of ET to PR230 (CMG2) cells. The cells were incubated with various concentrations of PA in the presence of EF (1 μg/mL) or LF (1 μg/mL) for 48 h, followed by an MTT assay to assess the cell viability. F-I. Cellular ATP depletion by ET vs. LT in multiple cell lines. MDA-MB-231 (F), B16-F10 (G), Glioma 261 (H) cells, and mouse primary fibroblasts (I) were incubated with ET (0.5 μg/mL or 5 μg/mL) or LT (5 μg/mL) for various lengths of time as indicated, followed by measuring cellular ATP levels. (A-I) Means ± SD of three independent biological replicates. J. The significant reduction in cellular ATP levels in livers from the mice treated with ET. Eighteen hours after challenge with 30 µg ET (I.V.), the mice were euthanized, and livers collected and processed for measuring tissue ATP levels. Means ± SD. Unpaired two-tailed Student’s t-test.
Fig 6.
ATP depletion by ET but not cholera toxin.
A. CHO cells were incubated with ET (PA/EF, each 2.5 µg/mL) for various lengths of time as indicated. Then cAMP (from conditioned medium, left panel) and cellular ATP (from cell lysates, middle panel) levels were assessed. Right panel, cell rounding morphological changes (at 24 h) were shown. ***, p < 0.01, when compared to the “0” groups. Unpaired two-tailed Student’s t-test. B, C. CHO cells were incubated with cholera toxin (5 µg/mL) (B) or forskolin (12.5 µM) (C) for various lengths of time as indicated. Then cAMP (from conditioned medium, left panels) and cellular ATP (from cell lysates, middle panels) levels were assessed. Right panels, cell rounding morphological changes (at 24 h) were shown. **, p < 0.05, ***, p < 0.01, when compared to the “0” groups. Unpaired two-tailed Student’s t-test. (A-C) Means ± SD of three independent biological replicates.