Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1975 Apr;11(4):665–674. doi: 10.1128/iai.11.4.665-674.1975

Chemical modulation of diphtheria toxin action on cultured mammalian cells.

B Ivins, C B Saelinger, P F Bonventre, C Woscinski
PMCID: PMC415119  PMID: 235491

Abstract

Ammonium chloride (4 times 10-3 M) rendered HEp-2 monolayers completely insensitive to the action of diphtheria toxin, as measured by de novo protein synthesis. Total protection was observed even with large amounts of toxin (400 minimum lethal doses/ml). Ammonium chloride did not reduce toxicity by direct action on the protein, nor did it prevent the adsorption of toxin to the cell membrane. Although the ammonium salt did not block the initial interaction between cell and toxin, it did maintain the toxin at a site amenable to neutralization with antitoxin. Surface-adsorbed toxin was inactivated by cellular enzymes or alternatively was desorbed from the membrane during a 12-h incubation in the presence of ammonium chloride. In addition, ammonium chloride provided protection to both toxin-sensitive guinea pig peritoneal macrophages and a partially toxin-resistant strain of HEp-2 cells. Sodium arsenite was effective in protecting cell monolayers from the action of diphtheria toxin; unlike ammonium chloride, its action was not dependent upon continued incubation with cells during exposure to toxin. Inhibitors of energy metabolism abolished toxin action either totally (sodium fluoride) or partially (dinitrophenol and sodium cyanide). Inhibitors of cellular proteases, on the other hand, did not modify toxin activity. The ability of several modifiers of membrane function to alter expression of toxicity for HEp-2 cells was also examined. One compound known to enhance endocytic activity, Tuftsin, had no effect, whereas poly-L-ornithine provided partial protection. Of the two compounds known to alter membrane fluidity, cytochalasin B provided partial protection for HEp-2 cell cultures, whereas colchicine had no effect. Agents that bind to sulfhydryl groups on the cell surface had no apparent effect on toxicity, suggesting that the initial toxin-cell interaction does not involve sulfhydryl groups. Those compounds that provide virtually full protection against the action of diphtheria toxic on cell monolayers (i.e., ammonium chloride, sodium fluoride, and sodium arsenite) had no inhibitory effect on the in vitro enzyme activity associated with fragment A of the toxin.

Full text

PDF
665

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BARKSDALE L., GARMISE L., HORIBATA K. Virulence, toxinogeny, and lysogeny in Corynebacterium diphtheriae. Ann N Y Acad Sci. 1960 Nov 21;88:1093–1108. doi: 10.1111/j.1749-6632.1960.tb20099.x. [DOI] [PubMed] [Google Scholar]
  2. Bonventre P. F., Imhoff J. G. Studies on the mode of action of diphtheria toxin. II. Protein synthesis in primary heart cell cultures. J Exp Med. 1967 Dec 1;126(6):1079–1086. doi: 10.1084/jem.126.6.1079. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bonventre P. F., Saelinger C. B., Imhoff J. G. Studies on the effect of diphtheria toxin on protein synthesis in mice. J Med Microbiol. 1973 May;6(2):169–176. doi: 10.1099/00222615-6-2-169. [DOI] [PubMed] [Google Scholar]
  4. Borisy G. G., Taylor E. W. The mechanism of action of colchicine. Binding of colchincine-3H to cellular protein. J Cell Biol. 1967 Aug;34(2):525–533. doi: 10.1083/jcb.34.2.525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cohn Z. A. The regulation of pinocytosis in mouse macrophages. I. Metabolic requirements as defined by the use of inhibitors. J Exp Med. 1966 Oct 1;124(4):557–571. doi: 10.1084/jem.124.4.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Collier R. J. Effect of diphtheria toxin on protein synthesis: inactivation of one of the transfer factors. J Mol Biol. 1967 Apr 14;25(1):83–98. doi: 10.1016/0022-2836(67)90280-x. [DOI] [PubMed] [Google Scholar]
  7. Collier R. J., Kandel J. Structure and activity of diphtheria toxin. I. Thiol-dependent dissociation of a fraction of toxin into enzymically active and inactive fragments. J Biol Chem. 1971 Mar 10;246(5):1496–1503. [PubMed] [Google Scholar]
  8. Constantopoulos A., Najjar V. A., Smith J. W. Tuftsin deficiency: a new syndrome with defective phagocytosis. J Pediatr. 1972 Apr;80(4):564–572. doi: 10.1016/s0022-3476(72)80051-9. [DOI] [PubMed] [Google Scholar]
  9. Drazin R., Kandel J., Collier R. J. Structure and activity of diphtheria toxin. II. Attack by trypsin at a specific site within the intact toxin molecule. J Biol Chem. 1971 Mar 10;246(5):1504–1510. [PubMed] [Google Scholar]
  10. Duncan J. L., Groman N. B. Activity of diphtheria toxin. II. Early events in the intoxication of HeLa cells. J Bacteriol. 1969 Jun;98(3):963–969. doi: 10.1128/jb.98.3.963-969.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gee J. B., Khandwala A. S., Bell R. W. Hexose transport in the alveolar macrophage: kinetics and pharmacologic features. J Reticuloendothel Soc. 1974 May;15(5):394–405. [PubMed] [Google Scholar]
  12. Gill D. M., Dinius L. L. Observations on the structure of diphtheria toxin. J Biol Chem. 1971 Mar 10;246(5):1485–1491. [PubMed] [Google Scholar]
  13. Gill D. M., Pappenheimer A. M., Jr Structure-activity relationships in diphtheria toxin. J Biol Chem. 1971 Mar 10;246(5):1492–1495. [PubMed] [Google Scholar]
  14. Honjo T., Nishizuka Y., Kato I., Hayaishi O. Adenosine diphosphate ribosylation of aminoacyl transferase II and inhibition of protein synthesis by diphtheria toxin. J Biol Chem. 1971 Jul 10;246(13):4251–4260. [PubMed] [Google Scholar]
  15. Ittelson T. R., Gill D. M. Diphtheria toxin: specific competition for cell receptors. Nature. 1973 Mar 30;242(5396):330–332. doi: 10.1038/242330b0. [DOI] [PubMed] [Google Scholar]
  16. Kim K., Groman N. B. In vitro inhibition of diphtheria toxin action by ammonium salts and amines. J Bacteriol. 1965 Dec;90(6):1552–1556. doi: 10.1128/jb.90.6.1552-1556.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kim K., Groman N. B. Mode of inhibition of diphtheria toxin by ammonium chloride. J Bacteriol. 1965 Dec;90(6):1557–1562. doi: 10.1128/jb.90.6.1557-1562.1965. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Liebow C., Rothman S. S. Membrane transport of proteins. Nat New Biol. 1972 Dec 6;240(101):176–178. doi: 10.1038/newbio240176a0. [DOI] [PubMed] [Google Scholar]
  19. Mehrishi J. N., Grassetti D. R. Sulphydryl groups on the surface of intact Ehrlich ascites tumour cells, human blood platelets and lymphocytes. Nature. 1969 Nov 8;224(5219):563–564. doi: 10.1038/224563a0. [DOI] [PubMed] [Google Scholar]
  20. Moehring J. M., Moehring T. J. The response of cultured mammalian cells to diphtheria toxin. II. The resistant cell: enhancement of toxin action by poly-L-ornithine. J Exp Med. 1968 Mar 1;127(3):541–554. doi: 10.1084/jem.127.3.541. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Moehring T. J., Moehring J. M. Response of cultured mammalian cells to diphtheria toxin. IV. Isolation of KB cells resistant to diphtheria toxin. Infect Immun. 1972 Oct;6(4):487–492. doi: 10.1128/iai.6.4.487-492.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. OYAMA V. I., EAGLE H. Measurement of cell growth in tissue culture with a phenol reagent (folin-ciocalteau). Proc Soc Exp Biol Med. 1956 Feb;91(2):305–307. doi: 10.3181/00379727-91-22245. [DOI] [PubMed] [Google Scholar]
  23. Tsan M. F., Berlin R. D. Effect of phagocytosis on membrane transport of nonelectrolytes. J Exp Med. 1971 Oct 1;134(4):1016–1035. doi: 10.1084/jem.134.4.1016. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Uchida T., Pappenheimer A. M., Jr, Harper A. A. Reconstitution of diphtheria toxin from two nontoxic cross-reacting mutant proteins. Science. 1972 Feb 25;175(4024):901–903. doi: 10.1126/science.175.4024.901. [DOI] [PubMed] [Google Scholar]
  25. Wessells N. K., Spooner B. S., Ash J. F., Bradley M. O., Luduena M. A., Taylor E. L., Wrenn J. T., Yamada K. Microfilaments in cellular and developmental processes. Science. 1971 Jan 15;171(3967):135–143. doi: 10.1126/science.171.3967.135. [DOI] [PubMed] [Google Scholar]
  26. Zigmond S. H., Hirsch J. G. Cytochalasin B: inhibition of D-2-deoxyglucose transport into leukocytes and fibroblasts. Science. 1972 Jun 30;176(4042):1432–1434. doi: 10.1126/science.176.4042.1432. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES