Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1996 Aug;64(8):2974–2979. doi: 10.1128/iai.64.8.2974-2979.1996

Selective killing of human monocytes and cytokine release provoked by sphingomyelinase (beta-toxin) of Staphylococcus aureus.

I Walev 1, U Weller 1, S Strauch 1, T Foster 1, S Bhakdi 1
PMCID: PMC174177  PMID: 8757823

Abstract

The best-known activity of Staphylococcus aureus sphingomyelinase C, alias beta-toxin, is as a hemolysin that provokes hot-cold lysis of erythrocytes which contain substantial amounts of sphingomyelin in the plasma membrane. Sheep erythrocytes are most susceptible, and we found that one hemolytic unit, representing the toxin concentration that elicits 50% hemolysis of 2.5 X 10(8) erythrocytes per ml, corresponds to 0.05 enzyme units or to approximately 0.25 microg of sphingomyelinase per ml. The cytotoxic action of beta-toxin on nucleated cells has not been described in any detail before, and the present investigation was undertaken to fill this information gap. We now identify beta-toxin as a remarkably potent monocytocidal agent. At a concentration of 0.001 U/ml, corresponding to approximately 5 ng/ml, beta-toxin killed over 50% of human monocytes (10(6) cells per ml) within 60 min. By contrast, 1 to 5 microg of beta-toxin per ml had no cytocidal effects on human granulocytes, fibroblasts, lymphocytes, or erythrocytes. A selective monocytocidal action was also observed with sphingomyelinase C from Bacillus cereus and a Streptomyces sp., whereas phospholipase A2 and phospholipase D at 100 U/ml were without effect. Monocytes succumbing to the action of beta-toxin processed and released interleukin-1beta, soluble interleukin-6 receptor, and soluble CD14 into the supernatant. Thus, monocyte killing by beta-toxin is associated with cytokine-related events that are important for the initiation and progression of infectious disease. These findings uncover a potentially important role for sphingomyelinase as a determinant of microbial pathogenicity.

Full Text

The Full Text of this article is available as a PDF (278.4 KB).

Selected References

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

  1. Bazil V. Physiological enzymatic cleavage of leukocyte membrane molecules. Immunol Today. 1995 Mar;16(3):135–140. doi: 10.1016/0167-5699(95)80130-8. [DOI] [PubMed] [Google Scholar]
  2. Bhakdi S., Greulich S., Muhly M., Eberspächer B., Becker H., Thiele A., Hugo F. Potent leukocidal action of Escherichia coli hemolysin mediated by permeabilization of target cell membranes. J Exp Med. 1989 Mar 1;169(3):737–754. doi: 10.1084/jem.169.3.737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bhakdi S., Grimminger F., Suttorp N., Walmrath D., Seeger W. Proteinaceous bacterial toxins and pathogenesis of sepsis syndrome and septic shock: the unknown connection. Med Microbiol Immunol. 1994 Jul;183(3):119–144. doi: 10.1007/BF00196048. [DOI] [PubMed] [Google Scholar]
  4. Bhakdi S., Muhly M., Korom S., Hugo F. Release of interleukin-1 beta associated with potent cytocidal action of staphylococcal alpha-toxin on human monocytes. Infect Immun. 1989 Nov;57(11):3512–3519. doi: 10.1128/iai.57.11.3512-3519.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bhakdi S., Muhly M., Korom S., Schmidt G. Effects of Escherichia coli hemolysin on human monocytes. Cytocidal action and stimulation of interleukin 1 release. J Clin Invest. 1990 Jun;85(6):1746–1753. doi: 10.1172/JCI114631. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bhakdi S., Weller U., Walev I., Martin E., Jonas D., Palmer M. A guide to the use of pore-forming toxins for controlled permeabilization of cell membranes. Med Microbiol Immunol. 1993 Sep;182(4):167–175. doi: 10.1007/BF00219946. [DOI] [PubMed] [Google Scholar]
  7. Bramley A. J., Patel A. H., O'Reilly M., Foster R., Foster T. J. Roles of alpha-toxin and beta-toxin in virulence of Staphylococcus aureus for the mouse mammary gland. Infect Immun. 1989 Aug;57(8):2489–2494. doi: 10.1128/iai.57.8.2489-2494.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Byers D. M., Morgan M. W., Cook H. W., Palmer F. B., Spence M. W. Niemann-Pick type II fibroblasts exhibit impaired cholesterol esterification in response to sphingomyelin hydrolysis. Biochim Biophys Acta. 1992 Jan 16;1138(1):20–26. doi: 10.1016/0925-4439(92)90146-e. [DOI] [PubMed] [Google Scholar]
  9. Coleman D. C., Arbuthnott J. P., Pomeroy H. M., Birkbeck T. H. Cloning and expression in Escherichia coli and Staphylococcus aureus of the beta-lysin determinant from Staphylococcus aureus: evidence that bacteriophage conversion of beta-lysin activity is caused by insertional inactivation of the beta-lysin determinant. Microb Pathog. 1986 Dec;1(6):549–564. doi: 10.1016/0882-4010(86)90040-9. [DOI] [PubMed] [Google Scholar]
  10. DOERY H. M., MAGNUSSON B. J., CHEYNE I. M., SULASEKHARAM J. A phospholipase in staphylococcal toxin which hydrolyses sphingomyelin. Nature. 1963 Jun 15;198:1091–1092. doi: 10.1038/1981091a0. [DOI] [PubMed] [Google Scholar]
  11. Denholm E. M., Wolber F. M. A simple method for the purification of human peripheral blood monocytes. A substitute for Sepracell-MN. J Immunol Methods. 1991 Nov 22;144(2):247–251. doi: 10.1016/0022-1759(91)90092-t. [DOI] [PubMed] [Google Scholar]
  12. Doery H. M., Magnusson B. J., Gulasekharam J., Pearson J. E. The properties of phospholipase enzymes in staphylococcal toxins. J Gen Microbiol. 1965 Aug;40(2):283–296. doi: 10.1099/00221287-40-2-283. [DOI] [PubMed] [Google Scholar]
  13. Gatt S., Dinur T., Barenholz Y. A spectrophotometric method for determination of sphingomyelinase. Biochim Biophys Acta. 1978 Sep 28;530(3):503–507. doi: 10.1016/0005-2760(78)90169-8. [DOI] [PubMed] [Google Scholar]
  14. Haimovitz-Friedman A., Kan C. C., Ehleiter D., Persaud R. S., McLoughlin M., Fuks Z., Kolesnick R. N. Ionizing radiation acts on cellular membranes to generate ceramide and initiate apoptosis. J Exp Med. 1994 Aug 1;180(2):525–535. doi: 10.1084/jem.180.2.525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hedström S. A., Malmqvist T. Sphingomyelinase activity of Staphylococcus aureus strains from recurrent furunculosis and other infections. Acta Pathol Microbiol Immunol Scand B. 1982 Jun;90(3):217–220. doi: 10.1111/j.1699-0463.1982.tb00108.x. [DOI] [PubMed] [Google Scholar]
  16. Johansen T., Holm T., Guddal P. H., Sletten K., Haugli F. B., Little C. Cloning and sequencing of the gene encoding the phosphatidylcholine-preferring phospholipase C of Bacillus cereus. Gene. 1988 May 30;65(2):293–304. doi: 10.1016/0378-1119(88)90466-0. [DOI] [PubMed] [Google Scholar]
  17. Jonas D., Walev I., Berger T., Liebetrau M., Palmer M., Bhakdi S. Novel path to apoptosis: small transmembrane pores created by staphylococcal alpha-toxin in T lymphocytes evoke internucleosomal DNA degradation. Infect Immun. 1994 Apr;62(4):1304–1312. doi: 10.1128/iai.62.4.1304-1312.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kennett F. F., Schenkein H. A., Ellis T. M., Rutherford R. B. Phospholipid composition of human monocytes and alterations occurring due to culture and stimulation by C3b. Biochim Biophys Acta. 1984 Jul 20;804(3):301–307. doi: 10.1016/0167-4889(84)90133-2. [DOI] [PubMed] [Google Scholar]
  19. Marinetti G. V., Cattieu K. Composition and metabolism of phospholipids of human leukocytes. Chem Phys Lipids. 1982 Oct;31(2):169–177. doi: 10.1016/0009-3084(82)90042-1. [DOI] [PubMed] [Google Scholar]
  20. Mohler K. M., Sleath P. R., Fitzner J. N., Cerretti D. P., Alderson M., Kerwar S. S., Torrance D. S., Otten-Evans C., Greenstreet T., Weerawarna K. Protection against a lethal dose of endotoxin by an inhibitor of tumour necrosis factor processing. Nature. 1994 Jul 21;370(6486):218–220. doi: 10.1038/370218a0. [DOI] [PubMed] [Google Scholar]
  21. Müllberg J., Durie F. H., Otten-Evans C., Alderson M. R., Rose-John S., Cosman D., Black R. A., Mohler K. M. A metalloprotease inhibitor blocks shedding of the IL-6 receptor and the p60 TNF receptor. J Immunol. 1995 Dec 1;155(11):5198–5205. [PubMed] [Google Scholar]
  22. Nakamura T., Abe A., Balazovich K. J., Wu D., Suchard S. J., Boxer L. A., Shayman J. A. Ceramide regulates oxidant release in adherent human neutrophils. J Biol Chem. 1994 Jul 15;269(28):18384–18389. [PubMed] [Google Scholar]
  23. Novick R. Properties of a cryptic high-frequency transducing phage in Staphylococcus aureus. Virology. 1967 Sep;33(1):155–166. doi: 10.1016/0042-6822(67)90105-5. [DOI] [PubMed] [Google Scholar]
  24. O'Reilly M., de Azavedo J. C., Kennedy S., Foster T. J. Inactivation of the alpha-haemolysin gene of Staphylococcus aureus 8325-4 by site-directed mutagenesis and studies on the expression of its haemolysins. Microb Pathog. 1986 Apr;1(2):125–138. doi: 10.1016/0882-4010(86)90015-x. [DOI] [PubMed] [Google Scholar]
  25. Obeid L. M., Linardic C. M., Karolak L. A., Hannun Y. A. Programmed cell death induced by ceramide. Science. 1993 Mar 19;259(5102):1769–1771. doi: 10.1126/science.8456305. [DOI] [PubMed] [Google Scholar]
  26. Projan S. J., Kornblum J., Kreiswirth B., Moghazeh S. L., Eisner W., Novick R. P. Nucleotide sequence: the beta-hemolysin gene of Staphylococcus aureus. Nucleic Acids Res. 1989 Apr 25;17(8):3305–3305. doi: 10.1093/nar/17.8.3305. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pushkareva M., Obeid L. M., Hannun Y. A. Ceramide: an endogenous regulator of apoptosis and growth suppression. Immunol Today. 1995 Jun;16(6):294–297. doi: 10.1016/0167-5699(95)80184-7. [DOI] [PubMed] [Google Scholar]
  28. Rose-John S., Heinrich P. C. Soluble receptors for cytokines and growth factors: generation and biological function. Biochem J. 1994 Jun 1;300(Pt 2):281–290. doi: 10.1042/bj3000281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Slotte J. P., Bierman E. L. Depletion of plasma-membrane sphingomyelin rapidly alters the distribution of cholesterol between plasma membranes and intracellular cholesterol pools in cultured fibroblasts. Biochem J. 1988 Mar 15;250(3):653–658. doi: 10.1042/bj2500653. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Thornberry N. A., Bull H. G., Calaycay J. R., Chapman K. T., Howard A. D., Kostura M. J., Miller D. K., Molineaux S. M., Weidner J. R., Aunins J. A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature. 1992 Apr 30;356(6372):768–774. doi: 10.1038/356768a0. [DOI] [PubMed] [Google Scholar]
  31. Titball R. W. Bacterial phospholipases C. Microbiol Rev. 1993 Jun;57(2):347–366. doi: 10.1128/mr.57.2.347-366.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wadström T., Möllby R. Some biological properties of purified staphylococcal haemolysins. Toxicon. 1972 Aug;10(5):511–519. doi: 10.1016/0041-0101(72)90177-8. [DOI] [PubMed] [Google Scholar]
  33. Walev I., Palmer M., Martin E., Jonas D., Weller U., Höhn-Bentz H., Husmann M., Bhakdi S. Recovery of human fibroblasts from attack by the pore-forming alpha-toxin of Staphylococcus aureus. Microb Pathog. 1994 Sep;17(3):187–201. doi: 10.1006/mpat.1994.1065. [DOI] [PubMed] [Google Scholar]
  34. Walev I., Reske K., Palmer M., Valeva A., Bhakdi S. Potassium-inhibited processing of IL-1 beta in human monocytes. EMBO J. 1995 Apr 18;14(8):1607–1614. doi: 10.1002/j.1460-2075.1995.tb07149.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wilkinson P. C. Inhibition of leukocyte locomotion and chemotaxis by lipid-specific bacterial toxins. Nature. 1975 Jun 5;255(5508):485–487. doi: 10.1038/255485a0. [DOI] [PubMed] [Google Scholar]
  36. Wong K., Li X. B., Hunchuk N. N-acetylsphingosine (C2-ceramide) inhibited neutrophil superoxide formation and calcium influx. J Biol Chem. 1995 Feb 17;270(7):3056–3062. doi: 10.1074/jbc.270.7.3056. [DOI] [PubMed] [Google Scholar]

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

RESOURCES