Bacillus anthracis, the causative agent of anthrax disease, is lethal owing to the actions of two exotoxins: anthrax lethal toxin (LT) and oedema toxin (ET). The key tissue targets responsible for the lethal effects of these toxins are unknown. Here we generated cell-type-specific anthrax toxin receptor capillary morphogenesis protein-2 (CMG2)-null mice and cell-type-specific CMG2-expressing mice and challenged them with the toxins. Our results show that lethality induced by LT and ET occurs through damage to distinct cell types; whereas targeting cardiomyocytes and vascular smooth muscle cells is required for LT-induced mortality, ET-induced lethality occurs mainly through its action in hepatocytes. Notably, and in contradiction to what has been previously postulated, targeting of endothelial cells by either toxin does not seem to contribute significantly to lethality. Our findings demonstrate that B. anthracis has evolved to use LT and ET to induce host lethality by coordinately damaging two distinct vital systems.
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This research was supported by the intramural research programs of the National Institute of Allergy and Infectious Diseases and the National Heart, Lung, and Blood Institute, National Institutes of Health. We thank L. Feigenbaum and the staff at SAIC/NCI Frederick for generation of the founder CMG2 transgenic mice. We thank A. Kulkarni, B. Hall, B. Klaunberg, S. Anderson, I. Sastalla, C. Leysath and C. Bachran for discussions, and D. Despres for help with echocardiography.
The authors declare no competing financial interests.
Extended data figures and tables
a, Strategy for generation of endothelial-cell-specific CMG2-null mice. Diagram shows CMG2fl allele having exon 12 (encoding transmembrane domain, TM) flanked by loxP sites and the endothelial-cell-specific CMG2-null allele (CMG2(EC)−). The red arrowheads indicate loxP sites. The homozygous endothelial-cell-specific CMG2-null mice (CMG2(EC)−/−) were obtained by intercrossing of CMG2(EC)+/−mice. Other cell-type-specific CMG2-null mice were made similarly by using the corresponding cell-type-specific Cre transgenic mice. b, RT–PCR analyses of CMG2 transmembrane (TM) domain deletion in various tissues of CMG2(EC)−/− mice. Primers flanking the CMG2 transmembrane domain were used to amplify a CMG2 cDNA fragment. Endothelial cells and non-endothelial cells were isolated simultaneously from lungs pooled from three CMG2(EC)−/− mice and three CMG2fl/fl control mice. Representative of two independent experiments is shown. Expression of TEM8 and GAPDH in these samples is also shown. c, Sensitivity of endothelial cells and non-endothelial cells from CMG2fl/fl and CMG2(EC)−/− mice to PA plus FP59. Cells were treated with various concentrations of PA and FP59 (100 ng ml−1) for 48 h. Cell viability was evaluated by MTT assay, expressed as relative MTT signals to untreated cells. Error bars indicate s.d. d, Resistance of endothelial cells from CMG2(EC)−/− mice to LT. Endothelial cells from CMG2(EC)−/− and wild-type mice were treated with various concentrations of LF and PA (500 ng ml−1) for 48 h. e, PA(L687A) preferentially kills CMG2-expressing cells. Cells were treated with various concentrations of PA or PA(L687A) and 100 ng ml−1 FP59 for 48 h. PR230(TEM8) and PR230(CMG2) are engineered CHO cells expressing only TEM8 or CMG2. Note that PR230(TEM8) cells are 100-fold more resistant than PR230(CMG2) cells to PA(L687A) plus FP59. f, Sensitivity of endothelial cells and non-endothelial cells from CMG2fl/fl and CMG2(EC)−/− mice to PA(L687A) plus FP59. Cells were incubated for 48 h with various concentrations of PA(L687A) and 100 ng ml−1 FP59. Error bars indicate s.d. g, Susceptibility of CMG2(EC)−/− mice to LT. CMG2(EC)−/− mice and their littermate controls were injected intravenously with 50 μg LT (50 μg PA plus 50 μg LF), and monitored for survival. Whole-body CMG2−/− mice were included as additional controls. h, Disease progression of the LT-challenged mice in panel g. Please see Methods for disease progression scoring criteria.
a, Strategy for generation of endothelial-cell-specific CMG2-expressing mice. In the CMG2 transgenic vector (LSL-CMG2), a loxP-stop-loxP cassette containing a promoterless eGFP and a poly(A) stop signal flanked by loxP sites was placed between the CAG promoter and CMG2 cDNA. Activation of CMG2 transgene in endothelial cells (CMG2EC) was achieved by breeding LSL-CMG2 mice with Cdh-cre mice to specifically remove the loxP-stop-loxP cassette in endothelial cells. Other cell-type-specific CMG2-expressing mice were made similarly by using the corresponding cell-type-specific Cre transgenic mice. b, c, Regained toxin sensitivity of endothelial cells from CMG2EC mice. Endothelial cells and non-endothelial cells from CMG2EC and whole-body CMG2−/− mice were incubated for 48 h with various concentrations of PA (b) or PA(L687A) (c) and 100 ng ml−1 FP59. Error bars indicate s.d.
a, RT–PCR analyses of CMG2 deletion in tissues of CMG2(CM )−/− and CMG2(SM/CM)−/− mice. CMG2 deletion was detected in the hearts of CMG2(CM)−/− mice and in the hearts and aorta of CMG2(SM/CM)−/− mice. The small fraction of CMG2 deletion that occurred in other tissues of the CMG2(SM/CM)−/− mice was due to the existence of varying amounts of vascular smooth muscle cells in those tissues. Representative of two independent experiments is shown. b, Resistance of smooth-muscle/cardiomyocyte-specific CMG2-null mice to LT. CMG2(SM/CM)−/− mice and their littermate CMG2(SM/CM)+/− controls were injected intravenously with 50 μg LT, and monitored for survival. Whole-body CMG2−/− mice were included as additional controls. Right panel shows the disease progression of the challenged mice. CMG2(SM/CM)−/− versus CMG2+/+ mice, P = 0.0002. Log-rank test.
a, Representative fluorescence microscopy of skeletal muscle (1), aorta (vascular smooth muscle) (2), small intestine (smooth muscle) (3), liver (4), lung (5), spleen (6) and kidney (cortex) (7) from CMG2CM mice (n = 2). Scale bar, 100 μm. b, Representative fluorescence microscopy of skeletal muscle (1), liver (2), kidney (cortex) (3), spleen (4) and uterus (5) from CMG2SM/CM mice (n = 3), and uterus (6) from LSL-CMG2 mice (n = 2). Scale bar, 100 μm.
a, Haematoxylin and eosin staining of heart and liver from wild-type (n = 3) and CMG2−/− (n = 3) mice challenged intraperitoneally with 100 μg LT for 48 h. In heart, regions with cardiomyocyte degeneration were found in LT-treated wild-type but not CMG2−/− mice. Arrows show examples of degenerated cardiomyocytes. In liver, regions with mild to modest hepatocyte degeneration were identified in LT-treated wild-type but not CMG2−/− mice. Arrows show examples of degenerated hepatocytes with cytosol vacuolization changes. Scale bar, 50 μm. b, Haematoxylin and eosin staining of heart and liver from wild-type (n = 4) and CMG2−/− (n = 3) mice 18 h after 50 μg ET injection (intravenously). In liver, regions with hepatocyte necrotic changes were identified in ET-treated wild-type mice but not CMG2−/− mice. Arrows show necrotic regions, arrowheads indicate examples of intact hepatocytes remaining in the necrotic regions. In heart, only scattered degenerated cardiomyocytes (arrow) were found in ET-treated wild-type but not CMG2−/− mice. Scale bar, 50 μm.
Extended Data Figure 6 Endothelial-cell-specific CMG2-null mice are sensitive to B. anthracis infection.
CMG2(EC)−/− mice and their littermate heterozygous mice were subcutaneously infected with 4 × 108 Sterne spores and monitored for survival. The right panel shows the disease progression of the challenged mice. Log-rank test.
a, Susceptibility of the hepatocyte-specific CMG2-null mice to LT. CMG2(Hep)−/− mice and their littermate CMG2+/+ control mice were challenged intraperitoneally with 100 μg LT and monitored for survival. Whole-body CMG2−/− mice were included as additional controls. b, Selective activation of CMG2 transgene in liver of CMG2Hep mice. Representative fluorescence microscopy of the liver (1), heart (3), skeletal muscles (4), lung (5), small intestines (smooth muscle) (6), aorta (7), spleen (8) and kidney (cortex) (9) from CMG2Hep mice (n = 2), and liver (2) from LSL-CMG2 mice (n = 2). Selective loss of GFP expression in liver from CMG2Hep mice but not LSL-CMG2 mice (1 and 2) is shown. Scale bar, 100 μm. c, Susceptibility of the hepatocyte-specific CMG2-expressing mice to LT. CMG2Hep mice and various control mice as indicated were intraperitoneally challenged with two doses of 100 μg LT and monitored for survival or signs of malaise. Right panel: disease progression of the challenged mice.
a, ET-induced footpad skin oedema in mice. Mice with various genotypes were injected with 0.25 μg ET (in 20 μl PBS) and the thicknesses of footpads were measured at 0, 8 and 20 h after injection. ET only induced modest oedema in CMG2−/− mice, but caused much higher levels of oedema in CMG2+/−, CMG2(EC)−/−, CMG2(SM/CM)−/− and CMG2(SM/CM/EC)−/− mice. The P values of the indicated groups versus CMG2+/− control group are shown. Each symbol represents one mouse. b–f, ET does not cause oedema in heart, spleen, kidney, lung and brain. Mice were challenged intravenously with 30 μg ET or PBS, and hearts (b), spleens (c), kidneys (d), brains (e) and lungs (f) were collected at 6 h or 18 h for tissue wet/dry ratio measurements. The P values of the indicated groups versus the PBS control group are shown. No significant differences were detected among the groups in b–e. In f, decreases in wet/dry ratio of lungs (dehydration) from ET-treated mice were observed. Each symbol represents one mouse. In a–e, error bars indicate s.e.; two-tailed unpaired t-test.
Extended Data Figure 9 Mice with CMG2 deletion in cardiovascular system and intestines remain sensitive to ET.
a, b, Sensitivity of CMG2(CM)−/−, CMG2(SM/CM/EC)−/−, CMG2(SM/CM/EC/IE)−/− and their littermate control mice to ET. Mice were challenged intravenously with 25 μg ET (a) or intraperitoneally with 50 μg ET (b) and survival monitored after challenge.
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Liu, S., Zhang, Y., Moayeri, M. et al. Key tissue targets responsible for anthrax-toxin-induced lethality. Nature 501, 63–68 (2013). https://doi.org/10.1038/nature12510
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