Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1995 Aug;63(8):3131–3136. doi: 10.1128/iai.63.8.3131-3136.1995

Cryptococcus neoformans melanin and virulence: mechanism of action.

Y Wang 1, P Aisen 1, A Casadevall 1
PMCID: PMC173427  PMID: 7622240

Abstract

Black melanin-like pigments are produced by several neurotropic fungi, including Cryptococcus neoformans. Pigment production is associated with virulence. In media containing phenolic substrates such as L-dopa, C. neoformans cells become black as a result of pigment accumulation. Pigmented and nonpigmented C. neoformans cells were studied with transmission electron microscopy and electron spin resonance (ESR) spectroscopy. Transmission electron microscopy showed electron-dense cell walls, and ESR spectroscopy revealed a stable free-radical population in pigmented cells. The ESR signals of pigmented cells were increased by light, alkaline pH, and Zn2+ and decreased by acid pH, indicating that the black pigment was a type of melanin. A mutant deficient in melanin synthesis (mel) generated by UV radiation lacked ESR-detectable radicals, was less virulent for mice, was more susceptible to killing by nitrogen- and oxygen-derived radicals, and had 100-foldless phenoloxidase activity than the parent strain. The interaction of melanized C. neoformans, nonmelanized C. neoformans, and the hypomelanotic mel mutant with J774.16 murine macrophage-like cells was studied. Melanized cells were more resistant to antibody-mediated phagocytosis and the antifungal effects of murine macrophages than nonmelanized cells. Small increases in the intensity of the ESR signals of melanized cells in solutions containing chemically generated oxygen- and nitrogen-derived radicals indicated electron transfer to or from melanin. Melanin appears to contribute to virulence by protecting fungal cells against attack by immune effector cells.

Full Text

The Full Text of this article is available as a PDF (531.5 KB).

Selected References

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

  1. Alspaugh J. A., Granger D. L. Inhibition of Cryptococcus neoformans replication by nitrogen oxides supports the role of these molecules as effectors of macrophage-mediated cytostasis. Infect Immun. 1991 Jul;59(7):2291–2296. doi: 10.1128/iai.59.7.2291-2296.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blasi E., Barluzzi R., Mazzolla R., Tancini B., Saleppico S., Puliti M., Pitzurra L., Bistoni F. Role of nitric oxide and melanogenesis in the accomplishment of anticryptococcal activity by the BV-2 microglial cell line. J Neuroimmunol. 1995 Apr;58(1):111–116. doi: 10.1016/0165-5728(95)00016-u. [DOI] [PubMed] [Google Scholar]
  3. Bustamante J., Bredeston L., Malanga G., Mordoh J. Role of melanin as a scavenger of active oxygen species. Pigment Cell Res. 1993 Oct;6(5):348–353. doi: 10.1111/j.1600-0749.1993.tb00612.x. [DOI] [PubMed] [Google Scholar]
  4. Casadevall A., Freundlich L. F., Marsh L., Scharff M. D. Extensive allelic variation in Cryptococcus neoformans. J Clin Microbiol. 1992 May;30(5):1080–1084. doi: 10.1128/jcm.30.5.1080-1084.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chan J., Fujiwara T., Brennan P., McNeil M., Turco S. J., Sibille J. C., Snapper M., Aisen P., Bloom B. R. Microbial glycolipids: possible virulence factors that scavenge oxygen radicals. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2453–2457. doi: 10.1073/pnas.86.7.2453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chan J., Xing Y., Magliozzo R. S., Bloom B. R. Killing of virulent Mycobacterium tuberculosis by reactive nitrogen intermediates produced by activated murine macrophages. J Exp Med. 1992 Apr 1;175(4):1111–1122. doi: 10.1084/jem.175.4.1111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dixon D. M., Polak A., Szaniszlo P. J. Pathogenicity and virulence of wild-type and melanin-deficient Wangiella dermatitidis. J Med Vet Mycol. 1987 Apr;25(2):97–106. doi: 10.1080/02681218780000141. [DOI] [PubMed] [Google Scholar]
  8. Eggleton P., Crawford N., Fisher D. Fractionation of human neutrophils into subpopulations by countercurrent distribution: surface charge and functional heterogeneity. Eur J Cell Biol. 1992 Apr;57(2):265–272. [PubMed] [Google Scholar]
  9. Enochs W. S., Nilges M. J., Swartz H. M. A standardized test for the identification and characterization of melanins using electron paramagnetic resonance (EPR) spectroscopy. Pigment Cell Res. 1993 Mar;6(2):91–99. doi: 10.1111/j.1600-0749.1993.tb00587.x. [DOI] [PubMed] [Google Scholar]
  10. Geis P. A., Wheeler M. H., Szaniszlo P. J. Pentaketide metabolites of melanin synthesis in the dematiaceous fungus Wangiella dermatitidis. Arch Microbiol. 1984 Apr;137(4):324–328. doi: 10.1007/BF00410729. [DOI] [PubMed] [Google Scholar]
  11. Granger D. L., Perfect J. R., Durack D. T. Macrophage-mediated fungistasis in vitro: requirements for intracellular and extracellular cytotoxicity. J Immunol. 1986 Jan;136(2):672–680. [PubMed] [Google Scholar]
  12. Hiemstra P. S., Eisenhauer P. B., Harwig S. S., van den Barselaar M. T., van Furth R., Lehrer R. I. Antimicrobial proteins of murine macrophages. Infect Immun. 1993 Jul;61(7):3038–3046. doi: 10.1128/iai.61.7.3038-3046.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hill H. Z. The function of melanin or six blind people examine an elephant. Bioessays. 1992 Jan;14(1):49–56. doi: 10.1002/bies.950140111. [DOI] [PubMed] [Google Scholar]
  14. Ikeda R., Jacobson E. S. Heterogeneity of phenol oxidases in Cryptococcus neoformans. Infect Immun. 1992 Sep;60(9):3552–3555. doi: 10.1128/iai.60.9.3552-3555.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Jacobson E. S., Tinnell S. B. Antioxidant function of fungal melanin. J Bacteriol. 1993 Nov;175(21):7102–7104. doi: 10.1128/jb.175.21.7102-7104.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kozel T. R. Dissociation of a hydrophobic surface from phagocytosis of encapsulated and non-encapsulated cryptococcus neoformans. Infect Immun. 1983 Mar;39(3):1214–1219. doi: 10.1128/iai.39.3.1214-1219.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kwon-Chung K. J., Hill W. B., Bennett J. E. New, special stain for histopathological diagnosis of cryptococcosis. J Clin Microbiol. 1981 Feb;13(2):383–387. doi: 10.1128/jcm.13.2.383-387.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kwon-Chung K. J., Polacheck I., Popkin T. J. Melanin-lacking mutants of Cryptococcus neoformans and their virulence for mice. J Bacteriol. 1982 Jun;150(3):1414–1421. doi: 10.1128/jb.150.3.1414-1421.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kwon-Chung K. J., Rhodes J. C. Encapsulation and melanin formation as indicators of virulence in Cryptococcus neoformans. Infect Immun. 1986 Jan;51(1):218–223. doi: 10.1128/iai.51.1.218-223.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. LONGUET-HIGGINS H. C. On the origin of the free radical property of melanins. Arch Biochem Biophys. 1960 Feb;86:231–232. doi: 10.1016/0003-9861(60)90410-0. [DOI] [PubMed] [Google Scholar]
  21. MASON H. S., INGRAM D. J., ALLEN B. The free radical property of melanins. Arch Biochem Biophys. 1960 Feb;86:225–230. doi: 10.1016/0003-9861(60)90409-4. [DOI] [PubMed] [Google Scholar]
  22. Mozaffarian N., Berman J. W., Casadevall A. Immune complexes increase nitric oxide production by interferon-gamma- stimulated murine macrophage-like J774.16 cells. J Leukoc Biol. 1995 Apr;57(4):657–662. doi: 10.1002/jlb.57.4.657. [DOI] [PubMed] [Google Scholar]
  23. Mukherjee S., Lee S. C., Casadevall A. Antibodies to Cryptococcus neoformans glucuronoxylomannan enhance antifungal activity of murine macrophages. Infect Immun. 1995 Feb;63(2):573–579. doi: 10.1128/iai.63.2.573-579.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mukherjee S., Lee S., Mukherjee J., Scharff M. D., Casadevall A. Monoclonal antibodies to Cryptococcus neoformans capsular polysaccharide modify the course of intravenous infection in mice. Infect Immun. 1994 Mar;62(3):1079–1088. doi: 10.1128/iai.62.3.1079-1088.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Perfect J. R., Magee B. B., Magee P. T. Separation of chromosomes of Cryptococcus neoformans by pulsed field gel electrophoresis. Infect Immun. 1989 Sep;57(9):2624–2627. doi: 10.1128/iai.57.9.2624-2627.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Polacheck I., Kwon-Chung K. J. Melanogenesis in Cryptococcus neoformans. J Gen Microbiol. 1988 Apr;134(4):1037–1041. doi: 10.1099/00221287-134-4-1037. [DOI] [PubMed] [Google Scholar]
  27. Polacheck I., Platt Y., Aronovitch J. Catecholamines and virulence of Cryptococcus neoformans. Infect Immun. 1990 Sep;58(9):2919–2922. doi: 10.1128/iai.58.9.2919-2922.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rhodes J. C., Polacheck I., Kwon-Chung K. J. Phenoloxidase activity and virulence in isogenic strains of Cryptococcus neoformans. Infect Immun. 1982 Jun;36(3):1175–1184. doi: 10.1128/iai.36.3.1175-1184.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Ro J. Y., Lee S. S., Ayala A. G. Advantage of Fontana-Masson stain in capsule-deficient cryptococcal infection. Arch Pathol Lab Med. 1987 Jan;111(1):53–57. [PubMed] [Google Scholar]
  30. Shaw C. E., Kapica L. Production of diagnostic pigment by phenoloxidase activity of cryptococcus neoformans. Appl Microbiol. 1972 Nov;24(5):824–830. doi: 10.1128/am.24.5.824-830.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. WHITE L. P. Melanin: a naturally occurring cation exchange material. Nature. 1958 Nov 22;182(4647):1427–1428. doi: 10.1038/1821427a0. [DOI] [PubMed] [Google Scholar]
  32. Walter H., Graham L. L., Krob E. J., Hill M. Correlation between phagocytic and membrane surface properties reflected by partitioning of human peripheral blood monocytes in two-polymer aqueous phases. Biochim Biophys Acta. 1980 Nov 4;602(2):309–322. doi: 10.1016/0005-2736(80)90314-4. [DOI] [PubMed] [Google Scholar]
  33. Wang Y., Casadevall A. Growth of Cryptococcus neoformans in presence of L-dopa decreases its susceptibility to amphotericin B. Antimicrob Agents Chemother. 1994 Nov;38(11):2648–2650. doi: 10.1128/aac.38.11.2648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wang Y., Casadevall A. Susceptibility of melanized and nonmelanized Cryptococcus neoformans to nitrogen- and oxygen-derived oxidants. Infect Immun. 1994 Jul;62(7):3004–3007. doi: 10.1128/iai.62.7.3004-3007.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Wheeler M. H., Bell A. A. Melanins and their importance in pathogenic fungi. Curr Top Med Mycol. 1988;2:338–387. doi: 10.1007/978-1-4612-3730-3_10. [DOI] [PubMed] [Google Scholar]
  36. Wheeler M. H., Stipanovic R. D. Melanin biosynthesis and the metabolism of flaviolin and 2-hydroxyjuglone in Wangiella dermatitidis. Arch Microbiol. 1985 Aug;142(3):234–241. doi: 10.1007/BF00693396. [DOI] [PubMed] [Google Scholar]
  37. Williamson P. R. Biochemical and molecular characterization of the diphenol oxidase of Cryptococcus neoformans: identification as a laccase. J Bacteriol. 1994 Feb;176(3):656–664. doi: 10.1128/jb.176.3.656-664.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Yoshida K., Akaike T., Doi T., Sato K., Ijiri S., Suga M., Ando M., Maeda H. Pronounced enhancement of .NO-dependent antimicrobial action by an .NO-oxidizing agent, imidazolineoxyl N-oxide. Infect Immun. 1993 Aug;61(8):3552–3555. doi: 10.1128/iai.61.8.3552-3555.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES