- Research Article
- Open Access
The cytotoxic effect of diphtheria toxin on the actin cytoskeleton
Cellular & Molecular Biology Letters volume 17, pages49–61(2012)
Diphtheria toxin (DT) and its N-terminal fragment A (FA) catalyse the transfer of the ADP-ribose moiety of nicotinamide adenine dinucleotide (NAD) into a covalent linkage with eukaryotic elongation factor 2 (eEF2). DT-induced cytotoxicity is versatile, and it includes DNA cleavage and the depolymerisation of actin filaments. The inhibition of the ADP-ribosyltransferase (ADPrT) activity of FA did not affect the deoxyribonuclease activity of FA or its interaction with actin. The toxin entry rate into cells (HUVEC) was determined by measuring the ADP-ribosyltransferase activity. DT uptake was nearly 80% after 30 min. The efficiency was determined as Km = 2.2 nM; Vmax = 0.25 pmol.min−1. The nuclease activity was tested with hyperchromicity experiments, and it was concluded that G-actin has an inhibitory effect on DT nuclease activity. In thepresence of DT and mutant of diphtheria toxin (CRM197), F-actin depolymerisation was determined with gel filtration, WB and fluorescence techniques. In the presence of DT and CRM197, 60–65% F-actin depolymerisation was observed. An in vitro FA-actin interaction and F-actin depolymerisation were reported in our previous paper. The present study thus confirms the depolymerisation of actin cytoskeleton in vivo.
globular actin monomer
human umbilical vein endothelial cells
Collier, R.J. Understanding the mode of action of diphtheria toxin: a perspective on progress during the 20th century. Toxicon 39 (2001) 1793–1803.
Choe, S., Bennett, M.J., Fujii, G., Curmi, P.M., Kantardjieff, K.A., Collier, R.J. and Eisenberg, D. The crystal structure of diphtheria toxin. Nature 357 (1994) 216–222.
Kageyama, T., Ohishi, M., Miyamoto, S., Mizushima, H., Iwamoto, R. and Mekada, E. Diphtheria toxin mutant CRM197 possesses weak EF2-ADPribosyl activity that potentiates its anti-tumorigenic activity. J. Biochem. 142 (2007) 95–104.
Rönnberg, B.J. and Middlebrook, J.L. Cellular regulation of diphtheria toxin cell surface receptors. Toxicon 27 (1989) 1377–1388.
Van Ness, B.G., Howard, J.B. and Bodley, J.W. ADP-ribosylation of elongation factor 2 by diphtheria toxin. NMR spectra and proposed structures of ribosyl-diphthamide and its hydrolysis products. J. Biochem. 255 (1980) 10710–10716.
Collier, R.J. Effect of diphtheria toxin on protein synthesis: inactivation of one of the transfer factors. J. Mol. Biol. 25 (1967) 83–98.
Draper, R.K. and Simon, M.I. The entry of diphtheria toxin into the mammalian cell cytoplasm: evidence for lysosomal involvement. J. Cell Biol. 87 (1980) 849–854.
Kaul, P., Silverman, J., Shen, W. H., Blanke, S.R., Huynh, P.D., Finkelstein, A. and Collier, J. Roles of Glu 349 and Asp 352 in membrane insertion and translocation by diphtheria toxin. Protein Sci. 5 (1996) 687–697.
D’silva, P.R. and Lala, A.K. Organisation of diphtheria toxin in membranes. A hydrophobic photolabeling study. J. Biol. Chem. 275 (1998) 11771–11777.
Lemichez, E., Bomsel, M., Devilliers, G., Vander Spek, J., Murphy, J.R., Lukianov, E.V., Olsnes, S. and Bouquet, P. Membrane translocation of diphtheria toxin fragment A exploits early to late endosome trafficking machinery. Mol. Microbiol. 23 (1997) 445–457.
Burch, G.E., Sun, S.C., Sohal, R.S., Chu, K.C and Colcolough, H.L. Diphtheritic myocarditis. A histochemical and electron microscopic study. Am. J. Cardiol. 21 (1968) 261–268.
Bektaş, M., Günçer, B., Güven, C., Nurten, R. and Bermek, E. Actin-an inhibitor of eukaryotic elongation factor activities. Biochem. Biophys. Res. Commun. 317 (2004) 1061–1066.
Bektaş, M., Varol, B., Nurten, R. and Bermek, E. Interaction of diphtheria toxin (fragment A) with actin. Cell Biochem. Funct. 27 (2009) 430–439.
Bektaş, M., Hacıosmanoğlu, E., Özerman, B., Varol, B., Nurten, R. and Bermek, E. The effect of cytochalasin D and the involvement of actin filaments and eukaryotic elongation factor 2 in the release of diphtheria toxin fragment a into the cytosol. Int. J. Biochem. Cell Biol. 43 (2011) 1365–1372.
Bektaş, M., Nurten, R., Ergen, K. and Bermek, E. Endogenous ADPribosylation for eukaryotic elongation factor 2: evidence of two different sites and reactions. Cell Biochem. Funct. 24 (2006) 369–380.
Bektaş., M., Nurten., R., Gürel., Z., Sayers, Z. and Bermek, E. Interactions of eukaryotic elongation factor 2 with actin: a possible link between protein synthetic machinery and cytoskeleton. FEBS Lett. 356 (1994) 89–93.
Bektaş, M., Nurten, R., Sayers, Z. and Bermek, E. Interactions of eukaryotic elongation factor 2 with the cytoskeleton interference with DNase I binding to actin. Eur. J. Biochem. 256 (1998) 142–147.
Engvall, E. Enzyme immunoassay ELISA and EMIT. Meth. Enzymol. 70 (1980) 419–439.
Laemmli, U.K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (1970) 680–685.
Dharmawardhane, S., Warren, V., Hall, A.L. and Condeelis, J. Changes in the assosication of actin binding proteins with the actin cytoskeleton during chemotactic stimulation of Dictiyostelium discoideum. Cell Motil. Cytoskeleton 13 (1989) 57–63.
Tu, Y., Wu, S., Shi, X., Chen, K. and Wu, C. Migfilin and Mig-2 ling focal adhesions to filamin and the actin cytoskeleton and function in cell shape modulation. Cell 113 (2003) 37–47.
Gibert, M., Marvaud, J.C., Pereira, Y., Hale, M.L., Stiles, B.G., Boquet, P., Lamaze, C. and Popoff. M.R. Differential requirement for the translocation of clostridial binary toxins: Iota toxin requires a membrane potential gradient. FEBS Lett. 581 (2007) 1287–1296.
Bruce, C., Baldvin, R.L., Lessnik, S.L. and Wisnieski, B.J. Diphtheria toxin and its ADP-ribosyltransferase-defective homologue CRM197 possess deoxyribonuclease activity. Proc. Natl. Acad. Sci. USA 87 (1990) 2995–2998.
Lessnick, S.L., Lyczak, J.B., Bruce, C., Lewis, D.G., Kim, P.S., Stolowitz, M.L., Hood, L. and Wisnieski, B.J. Localization of diphtheria toxin nuclease activity to fragment A. J. Bact. 174 (1992) 2032–2038.
Pappenheimer, A.M. Jr. and Gill, D.M. Diphtheria. Science 26 (1973) 353–358.
Collier, R.J. Diphtheria toxin: mode of action and structure. Bacteriol. Rev. 39 (1975) 54–85.
Morris, R.E. and Saelinger, C.E. Diphtheria toxin does not enter resistant cells by receptor-mediated endocytosis. Infect. Immun. 42 (1987) 812–817.
Bras, M., Queenan, B. and Susin, A. Programmed cell death via mitochondria: different modes of dying. Biochemistry (Mosc.) 70 (2005) 231–233.
Kusano, I., Kageyama, A., Tamura, T., Oda, T. and Muramatsu, T. Enhancement of diphtheria toxin-induced apoptosis in Vero cells by combination treatment with brefeldin A and okadaic acid. Cell Struct. Funct. 26 (2001) 279–288.
Barth, H., Olenik, C., Sehr, P., Schmidt, G., Aktories, K. and Mayer, D.K. Neosynthesis and activation of Rho by Escherichia coli cytotoxic necrotizing factor (CNF1) reverse cytopathic effects of ADP-ribosylated Rho. J. Biol. Chem. 274 (1999) 27407–27414.
Cha, J.H., Brook, J.S., Ivey, K.N. and Eidels, L. Cell surface monkey CD9 antigen is a coreceptor that increases diphtheria toxin sensitivity and diphtheria toxin receptor affinity. J. Biol Chem. 275 (2000) 6901–6907.
Brooke, J.S., Cha, J.H. and Eidels, L. Diphtheria toxin receptor interaction: association, dissociation, and effect of pH. Biochem. Biophys. Res. Commun. 248 (1998) 297–302.
Richard, J.F., Petit, L., Gibert, M., Marvaud, J.C., Bouchaud, C. and Popoff, M. Bacterial toxins modifying the actin cytoskeleton. Internal. Microbiol. 2 (1999) 185–194.
Aktories, K. and Wegner, A. ADP-ribosylation of actin by clostridial toxins. J. Cell. Biol. 109 (1989) 1385–1387.
Aktories K., Barmann, M., Ohishi, I., Tsuyama, S., Jakobs, K.H. and Haberman, E. Botulinum C2 toxin ADP-ribosylates actin. Nature 322 (1986) 390–392.
Van den Ent, F., Amos, L.A. and Löwe, J. Prokaryotic origin of the actin cytoskeleton. Nature 413 (2001) 39–44.
About this article
Cite this article
Varol, B., Bektaş, M., Nurten, R. et al. The cytotoxic effect of diphtheria toxin on the actin cytoskeleton. Cell Mol Biol Lett 17, 49–61 (2012). https://doi.org/10.2478/s11658-011-0036-6
- Diphtheria toxin
- Eukaryotic elongation factor-2
- Fragment A