AMPD3 is involved in anthrax LeTx-induced macrophage cell death

Abstract

The responses of macrophages to Bacillus anthracis infection are important for the survival of the host, since macrophages are required for the germination of B. anthracis spores in lymph nodes, and macrophage death exacerbates anthrax lethal toxin (LeTx)-induced organ collapse. To elucidate the mechanism of macrophage cell death induced by LeTx, we performed a genetic screen to search for genes associated with LeTx-induced macrophage cell death. RAW 264.7 cells, a macrophage-like cell line sensitive to LeTx-induced death, were randomly mutated and LeTx-resistant mutant clones were selected. AMP deaminase 3 (AMPD3), an enzyme that converts AMP to IMP, was identified to be mutated in one of the resistant clones. The requirement of AMPD3 in LeTx-induced cell death of RAW 264.7 cells was confirmed by the restoration of LeTx sensitivity with ectopic reconstitution of AMPD3 expression. AMPD3 deficiency does not affect LeTx entering cells and the cleavage of mitogen-activated protein kinase kinase (MKK) by lethal factor inside cells, but does impair an unknown downstream event that is linked to cell death. Our data provides new information regarding LeTx-induced macrophage death and suggests that there is a key regulatory site downstream of or parallel to MKK cleavage that controls the cell death in LeTx-treated macrophages.

Keywords

AMP deaminase 3 / anthrax lethal toxin / macrophage / cell death

References

[1] Banks, D.J., Barnajian, M., Maldonado-Arocho, F.J., Sanchez, A.M., and Bradley, K.A. (2005). Anthrax toxin receptor 2 mediates Bacillus anthracis killing of macrophages following spore challenge. Cell Microbiol 7, 1173–1185 10.1111/j.1462-5822.2005.00545.x.
[2] Bazan, J.F., and Koch-Nolte, F. (1997). Sequence and structural links between distant ADP-ribosyltransferase families. Adv Exp Med Biol 419, 99–107 10.1007/978-1-4419-8632-0_12.
[3] Duesbery, N.S., Webb, C.P., Leppla, S.H., Gordon, V.M., Klimpel, K.R., Copeland, T.D., (1998). Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor. Science 280, 734–737 10.1126/science.280.5364.734.
[4] Friedlander, A.M. (1986). Macrophages are sensitive to anthrax lethal toxin through an acid-dependent process. J Biol Chem 261, 7123–7126 .
[5] Friedlander, A.M., Bhatnagar, R., Leppla, S.H., Johnson, L., and Singh, Y. (1993). Characterization of macrophage sensitivity and resistance to anthrax lethal toxin. Infect Immun 61, 245–252 .
[6] Guidi-Rontani, C., Levy, M., Ohayon, H., and Mock, M. (2001). Fate of germinated Bacillus anthracis spores in primary murine macrophages. Mol Microbiol 42, 931–938 10.1046/j.1365-2958.2001.02695.x.
[7] Kim, S.O., Ha, S.D., Lee, S., Stanton, S., Beutler, B., and Han, J. (2007). Mutagenesis by retroviral insertion in chemical mutagen-generated quasi-haploid mammalian cells. Biotechniques 42, 493–501 10.2144/000112390.
[8] Kim, S.O., Jing, Q., Hoebe, K., Beutler, B., Duesbery, N.S., and Han, J. (2003). Sensitizing anthrax lethal toxin-resistant macrophages to lethal toxin-induced killing by tumor necrosis factor-alpha. J Biol Chem 278, 7413–7421 10.1074/jbc.M209279200.
[9] McAllister, R.D., Singh, Y., Du Bois, W.D., Potter, M., Boehm, T., Meeker, N.D., Fillmore, P.D., Anderson, L.M., Poynter, M.E., and Teuscher, C. (2003). Susceptibility to anthrax lethal toxin is controlled by three linked quantitative trait loci. Am J Pathol 163, 1735–1741 10.1016/S0002-9440(10)63532-8.
[10] Moayeri, M., Haines, D., Young, H.A., and Leppla, S.H. (2003). Bacillus anthracis lethal toxin induces TNF-independent hypoxia-mediated toxicity in mice. J Clin Invest 112, 670–682 .
[11] Moayeri, M., and Leppla, S.H. (2009). Cellular and systemic effects of anthrax lethal toxin and edema toxin. Mol Aspects Med 30, 439–455 10.1016/j.mam.2009.07.003.
[12] Moayeri, M., Martinez, N.W., Wiggins, J., Young, H.A., and Leppla, S.H. (2004). Mouse susceptibility to anthrax lethal toxin is influenced by genetic factors in addition to those controlling macrophage sensitivity. Infect Immun 72, 4439–4447 10.1128/IAI.72.8.4439-4447.2004.
[13] Moayeri, M., Webster, J.I., Wiggins, J.F., Leppla, S.H., and Sternberg, E.M. (2005). Endocrine perturbation increases susceptibility of mice to anthrax lethal toxin. Infect Immun 73, 4238–4244 10.1128/IAI.73.7.4238-4244.2005.
[14] Morisaki, H., and Morisaki, T. (2008). AMPD genes and urate metabolism. Nippon Rinsho 66, 771–777 .
[15] Muehlbauer, S.M., Evering, T.H., Bonuccelli, G., Squires, R.C., Ashton, A.W., Porcelli, S.A., Lisanti, M.P., and Brojatsch, J. (2007). Anthrax lethal toxin kills macrophages in a strain-specific manner by apoptosis or caspase-1-mediated necrosis. Cell Cycle 6, 758–766 10.4161/cc.6.6.3991.
[16] Park, J.M., Greten, F.R., Li, Z.W., and Karin, M. (2002). Macrophage apoptosis by anthrax lethal factor through p38 MAP kinase inhibition. Science 297, 2048–2051 10.1126/science.1073163.
[17] Pellizzari, R., Guidi-Rontani, C., Vitale, G., Mock, M., and Montecucco, C. (1999). Anthrax lethal factor cleaves MKK3 in macrophages and inhibits the LPS/IFNgamma-induced release of NO and TNFalpha. FEBS Lett 462, 199–204 10.1016/S0014-5793(99)01502-1.
[18] Scobie, H.M., and Young, J.A. (2005). Interactions between anthrax toxin receptors and protective antigen. Curr Opin Microbiol 8, 106–112 10.1016/j.mib.2004.12.005.
[19] Smith, H. (2000). Discovery of the anthrax toxin: the beginning of in vivo studies on pathogenic bacteria. Trends Microbiol 8, 199–200 10.1016/S0966-842X(00)01755-8.
[20] Smith, H. (2002). Discovery of the anthrax toxin: the beginning of studies of virulence determinants regulated in vivo. Int J Med Microbiol 291, 411–417 10.1078/1438-4221-00147.
[21] Smith, H., and Keppie, J. (1954). Observations on experimental anthrax: demonstration of a specific lethal factor produced in vivo by Bacillus anthracis. Nature 173, 869–870 10.1038/173869a0.
[22] Vitale, G., Bernardi, L., Napolitani, G., Mock, M., and Montecucco, C. (2000). Susceptibility of mitogen-activated protein kinase kinase family members to proteolysis by anthrax lethal factor. Biochem J 352, 739–745 10.1042/0264-6021:3520739.
[23] Vitale, G., Pellizzari, R., Recchi, C., Napolitani, G., Mock, M., and Montecucco, C. (1998). Anthrax lethal factor cleaves the N-terminus of MAPKKs and induces tyrosine/threonine phosphorylation of MAPKs in cultured macrophages. Biochem Biophys Res Commun 248, 706–711 10.1006/bbrc.1998.9040.
[24] Wang, X., Ono, K., Kim, S.O., Kravchenko, V., Lin, S.C., and Han, J. (2001). Metaxin is required for tumor necrosis factorinduced cell death. EMBO Rep 2, 628–633 10.1093/embo-reports/kve135.