1887

Abstract

Melanization is an intrinsic characteristic of many fungal species, but details of this process are poorly understood because melanins are notoriously difficult pigments to study. While studying the binding of cell-wall dyes, Eosin Y or Uvitex, to melanized and non-melanized Cryptococcus neoformans cells we noted that melanization leads to reduced fluorescence intensity, suggesting that melanin interfered with dye binding to the cell wall. The growth of C. neoformans in melanizing conditions with either of the cell-wall dyes resulted in an increase in supernatant-associated melanin, consistent with blockage of melanin attachment to the cell wall. This effect provided the opportunity to characterize melanin released into culture supernatants. Released melanin particles appeared mostly as networked structures having dimensions consistent with previously described extracellular vesicles. Hence, dye binding to the cell wall created conditions that resembled the ‘leaky melanin’ phenotype described for certain cell-wall mutants. In agreement with earlier studies on fungal melanins biosynthesis, our observations are supportive of a model whereby C. neoformans melanization proceeds by the attachment of melanin nanoparticles to the cell wall through chitin, chitosan, and various glucans.

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2018-06-25
2024-04-23
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References

  1. Chen Z, Nunes MA, Silva MC, Rodrigues CJ. Appressorium turgor pressure of Colletotrichum kahawae might have a role in coffee cuticle penetration. Mycologia 2004; 96:1199–1208 [View Article][PubMed]
    [Google Scholar]
  2. Ál R, Casadevall A. Melanization affects susceptibility of Cryptococcus neoformans to heat and cold1. FEMS Microbiol Lett 2006; 153:265–272
    [Google Scholar]
  3. Wang Y, Casadevall A. Decreased susceptibility of melanized Cryptococcus neoformans to UV light. Appl Environ Microbiol 1994; 60:3864–3866[PubMed]
    [Google Scholar]
  4. Steenbergen JN, Shuman HA, Casadevall A. Cryptococcus neoformans interactions with amoebae suggest an explanation for its virulence and intracellular pathogenic strategy in macrophages. Proc Natl Acad Sci USA 2001; 98:15245–15250 [View Article][PubMed]
    [Google Scholar]
  5. Casadevall A, Cordero RJB, Bryan R, Nosanchuk J, Dadachova E. Melanin, radiation, and energy transduction in fungi. Microbiol Spectr 2017; 5: [View Article][PubMed]
    [Google Scholar]
  6. Jacobson ES. Pathogenic roles for fungal melanins. Clin Microbiol Rev 2000; 13:708–717 [View Article][PubMed]
    [Google Scholar]
  7. Ikeda R, Sugita T, Jacobson ES, Shinoda T. Effects of melanin upon susceptibility of Cryptococcus to antifungals. Microbiol Immunol 2003; 47:271–277 [View Article][PubMed]
    [Google Scholar]
  8. van Duin D, Casadevall A, Nosanchuk JD. Melanization of Cryptococcus neoformans and Histoplasma capsulatum reduces their susceptibilities to amphotericin B and caspofungin. Antimicrob Agents Chemother 2002; 46:3394–3400 [View Article][PubMed]
    [Google Scholar]
  9. Nosanchuk JD, Ovalle R, Casadevall A. Glyphosate inhibits melanization of Cryptococcus neoformans and prolongs survival of mice after systemic infection. J Infect Dis 2001; 183:1093–1099 [View Article][PubMed]
    [Google Scholar]
  10. Coelho C, Bocca AL, Casadevall A. The tools for virulence of Cryptococcus neoformans. Adv Appl Microbiol 2014; 87:1–41 [View Article][PubMed]
    [Google Scholar]
  11. Zhu X, Gibbons J, Garcia-Rivera J, Casadevall A, Williamson PR. Laccase of Cryptococcus neoformans is a cell wall-associated virulence factor. Infect Immun 2001; 69:5589–5596 [View Article][PubMed]
    [Google Scholar]
  12. Wang Y, Aisen P, Casadevall A. Cryptococcus neoformans melanin and virulence: mechanism of action. Infect Immun 1995; 63:3131–3136[PubMed]
    [Google Scholar]
  13. Eisenman HC, Frases S, Nicola AM, Rodrigues ML, Casadevall A. Vesicle-associated melanization in Cryptococcus neoformans. Microbiology 2009; 155:3860–3867 [View Article][PubMed]
    [Google Scholar]
  14. Rodrigues ML, Nakayasu ES, Oliveira DL, Nimrichter L, Nosanchuk JD et al. Extracellular vesicles produced by Cryptococcus neoformans contain protein components associated with virulence. Eukaryot Cell 2008; 7:58–67 [View Article][PubMed]
    [Google Scholar]
  15. Franzen AJ, Cunha MM, Miranda K, Hentschel J, Plattner H et al. Ultrastructural characterization of melanosomes of the human pathogenic fungus Fonsecaea pedrosoi. J Struct Biol 2008; 162:75–84 [View Article][PubMed]
    [Google Scholar]
  16. Zhong J, Frases S, Wang H, Casadevall A, Stark RE. Following fungal melanin biosynthesis with solid-state NMR: biopolymer molecular structures and possible connections to cell-wall polysaccharides. Biochemistry 2008; 47:4701–4710 [View Article][PubMed]
    [Google Scholar]
  17. Banks IR, Specht CA, Donlin MJ, Gerik KJ, Levitz SM et al. A chitin synthase and its regulator protein are critical for chitosan production and growth of the fungal pathogen Cryptococcus neoformans. Eukaryot Cell 2005; 4:1902–1912 [View Article][PubMed]
    [Google Scholar]
  18. Baker LG, Specht CA, Donlin MJ, Lodge JK. Chitosan, the deacetylated form of chitin, is necessary for cell wall integrity in Cryptococcus neoformans. Eukaryot Cell 2007; 6:855–867 [View Article][PubMed]
    [Google Scholar]
  19. Camacho E, Chrissian C, Cordero RJB, Liporagi-Lopes L, Stark RE et al. N-acetylglucosamine affects Cryptococcus neoformans cell-wall composition and melanin architecture. Microbiology 2017; 163:1540–1556 [View Article][PubMed]
    [Google Scholar]
  20. Welch AZ, Koshland DE. A simple colony-formation assay in liquid medium, termed 'tadpoling', provides a sensitive measure of Saccharomyces cerevisiae culture viability. Yeast 2013; 30:501–509 [View Article][PubMed]
    [Google Scholar]
  21. Riesz JJ. The Spectroscopic Properties of Melanin. PhD Thesi University of Queensland, Australia: 2007
    [Google Scholar]
  22. Eisenman HC, Mues M, Weber SE, Frases S, Chaskes S et al. Cryptococcus neoformans laccase catalyses melanin synthesis from both D- and L-DOPA. Microbiology 2007; 153:3954–3962 [View Article][PubMed]
    [Google Scholar]
  23. Rodrigues ML, Nimrichter L, Oliveira DL, Frases S, Miranda K et al. Vesicular polysaccharide export in Cryptococcus neoformans is a eukaryotic solution to the problem of fungal trans-cell wall transport. Eukaryot Cell 2007; 6:48–59 [View Article][PubMed]
    [Google Scholar]
  24. Bourbonnais R, Leech D, Paice MG. Electrochemical analysis of the interactions of laccase mediators with lignin model compounds. Biochim Biophys Acta 1998; 1379:381–390 [View Article][PubMed]
    [Google Scholar]
  25. Eisenman HC, Nosanchuk JD, Webber JB, Emerson RJ, Camesano TA et al. Microstructure of cell wall-associated melanin in the human pathogenic fungus Cryptococcus neoformans. Biochemistry 2005; 44:3683–3693 [View Article][PubMed]
    [Google Scholar]
  26. Coleman T, Madassery JV, Kobayashi GS, Nahm MH, Little JR. New fluorescence assay for the quantitation of fungi. J Clin Microbiol 1989; 27:2003–2007[PubMed]
    [Google Scholar]
  27. Walton FJ, Idnurm A, Heitman J. Novel gene functions required for melanization of the human pathogen Cryptococcus neoformans. Mol Microbiol 2005; 57:1381–1396 [View Article][PubMed]
    [Google Scholar]
  28. Bull AT. Chemical composition of wild-type and mutant Aspergillus nidulans cell walls. The nature of polysaccharide and melanin constituents. J Gen Microbiol 1970; 63:75–94 [View Article][PubMed]
    [Google Scholar]
  29. Wang Z, Szaniszlo PJ. WdCHS3, a gene that encodes a class III chitin synthase in Wangiella (Exophiala) dermatitidis, is expressed differentially under stress conditions. J Bacteriol 2000; 182:874–881 [View Article][PubMed]
    [Google Scholar]
  30. Walker CA, Gómez BL, Mora-Montes HM, MacKenzie KS, Munro CA et al. Melanin externalization in Candida albicans depends on cell wall chitin structures. Eukaryot Cell 2010; 9:1329–1342 [View Article][PubMed]
    [Google Scholar]
  31. Heustis RJ, Ng HK, Brand KJ, Rogers MC, Le LT et al. Pharyngeal polysaccharide deacetylases affect development in the nematode C. elegans and deacetylate chitin in vitro. PLoS One 2012; 7:e40426 [View Article][PubMed]
    [Google Scholar]
  32. Zheng W, Fan H, Wang L, Jin Z. Oxidative self-polymerization of dopamine in an acidic environment. Langmuir 2015; 31:11671–11677 [View Article][PubMed]
    [Google Scholar]
  33. Roncero C, Durán A. Effect of Calcofluor white and Congo red on fungal cell wall morphogenesis: in vivo activation of chitin polymerization. J Bacteriol 1985; 163:1180–1185[PubMed]
    [Google Scholar]
  34. Hernández C, Farnet da Silva AM, Ziarelli F, Perraud-Gaime I, Gutiérrez-Rivera B et al. Laccase induction by synthetic dyes in Pycnoporus sanguineus and their possible use for sugar cane bagasse delignification. Appl Microbiol Biotechnol 2017; 101:1189–1201 [View Article][PubMed]
    [Google Scholar]
  35. Nar G, Latge J-P, Munro CA. The fungal cell wall: structure, biosynthesis, and function. Microbiol Spectr 2017; 5:
    [Google Scholar]
  36. Walker L, Sood P, Lenardon MD, Milne G, Olson J et al. The viscoelastic properties of the fungal cell wall allow traffic of AmBisome as intact liposome vesicles. MBio 2018; 9:e02383-17 [View Article][PubMed]
    [Google Scholar]
  37. Wang Z, Zheng L, Hauser M, Becker JM, Szaniszlo PJ. WdChs4p, a homolog of chitin synthase 3 in Saccharomyces cerevisiae, alone cannot support growth of Wangiella (Exophiala) dermatitidis at the temperature of infection. Infect Immun 1999; 67:6619–6630[PubMed]
    [Google Scholar]
  38. 25 Maeda H, Ishida N. Specificity of binding of hexopyranosyl polysaccharides with fluorescent brightener. J Biochem 1967; 62:276–278 [View Article][PubMed]
    [Google Scholar]
  39. Wachsmuth ED. A comparison of the highly selective fluorescence staining of fungi in tissue sections with Uvitex 2B and Calcofluor White M2R. Histochem J 1988; 20:215–221 [View Article][PubMed]
    [Google Scholar]
  40. Zhang P, Wei D, Li Z, Sun Z, Pan J et al. Cryptococcal phosphoglucose isomerase is required for virulence factor production, cell wall integrity and stress resistance. FEMS Yeast Res 2015; 15:fov072 [View Article][PubMed]
    [Google Scholar]
  41. Tsirilakis K, Kim C, Vicencio AG, Andrade C, Casadevall A et al. Methylxanthine inhibit fungal chitinases and exhibit antifungal activity. Mycopathologia 2012; 173:83–91 [View Article][PubMed]
    [Google Scholar]
  42. Wolf JM, Espadas-Moreno J, Luque-Garcia JL, Casadevall A. Interaction of Cryptococcus neoformans extracellular vesicles with the cell wall. Eukaryot Cell 2014; 13:1484–1493 [View Article][PubMed]
    [Google Scholar]
  43. Mandal P, Roy TS, das TK, Banerjee U, Xess I et al. Differences in the cell wall architecture of melanin lacking and melanin producing Cryptococcus neoformans clinical isolates from India: an electron microscopic study. Braz J Microbiol 2007; 38:662–666 [View Article]
    [Google Scholar]
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