1887

Abstract

is an environmental fungus that belongs to the phylum and is a major pathogen in immunocompromised patients. The ability of to produce melanin pigments represents its second most important virulence factor, after the presence of a polysaccharide capsule. Both the capsule and melanin are closely associated with the fungal cell wall, a complex structure that is essential for maintaining cell morphology and viability under conditions of stress. The amino sugar -acetylglucosamine (GlcNAc) is a key constituent of the cell-wall chitin and is used for both N-linked glycosylation and GPI anchor synthesis. Recent studies have suggested additional roles for GlcNAc as an activator and mediator of cellular signalling in fungal and plant cells. Furthermore, chitin and chitosan polysaccharides interact with melanin pigments in the cell wall and have been found to be essential for melanization. Despite the importance of melanin, its molecular structure remains unresolved; however, we previously obtained critical insights using advanced nuclear magnetic resonance (NMR) and imaging techniques. In this study, we investigated the effect of GlcNAc supplementation on cryptococcal cell-wall composition and melanization. was able to metabolize GlcNAc as a sole source of carbon and nitrogen, indicating a capacity to use a component of a highly abundant polymer in the biospherenutritionally. cells grown with GlcNAc manifested changes in the chitosan cell-wall content, cell-wall thickness and capsule size. Supplementing cultures with isotopically N-labelled GlcNAc demonstrated that the exogenous monomer serves as a building block for chitin/chitosan and is incorporated into the cell wall. The altered chitin-to-chitosan ratio had no negative effects on the mother–daughter cell separation; growth with GlcNAc affected the fungal cell-wall scaffold, resulting in increased melanin deposition and assembly. In summary, GlcNAc supplementation had pleiotropic effects on cell-wall and melanin architectures, and thus established its capacity to perturb these structures, a property that could prove useful for metabolic tracking studies.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000552
2017-11-01
2020-02-17
Loading full text...

Full text loading...

/deliver/fulltext/micro/163/11/1540.html?itemId=/content/journal/micro/10.1099/mic.0.000552&mimeType=html&fmt=ahah

References

  1. Casadevall A, Perfect JR. Cryptococcus Neoformans Washington, DC: American Society for Microbiology; 1998;[Crossref]
    [Google Scholar]
  2. Meya D, Rajasingham R, Nalintya E, Tenforde M, Jarvis JN. Preventing cryptococcosis-shifting the paradigm in the era of highly active antiretroviral therapy. Curr Trop Med Rep 2015;2:81–89 [CrossRef][PubMed]
    [Google Scholar]
  3. Mirza SA, Phelan M, Rimland D, Graviss E, Hamill R et al. The changing epidemiology of cryptococcosis: an update from population-based active surveillance in 2 large metropolitan areas, 1992–2000. Clin Infect Dis 2003;36:789–794 [CrossRef][PubMed]
    [Google Scholar]
  4. Alspaugh JA. Virulence mechanisms and Cryptococcus neoformans pathogenesis. Fungal Genet Biol 2015;78:55–58 [CrossRef][PubMed]
    [Google Scholar]
  5. McClelland EE, Bernhardt P, Casadevall A. Estimating the relative contributions of virulence factors for pathogenic microbes. Infect Immun 2006;74:1500–1504 [CrossRef][PubMed]
    [Google Scholar]
  6. Cherniak R, Sundstrom JB. Polysaccharide antigens of the capsule of Cryptococcus neoformans. Infect Immun 1994;62:1507–1512[PubMed]
    [Google Scholar]
  7. Reese AJ, Doering TL. Cell wall alpha-1,3-glucan is required to anchor the Cryptococcus neoformans capsule. Mol Microbiol 2003;50:1401–1409 [CrossRef][PubMed]
    [Google Scholar]
  8. Reese AJ, Yoneda A, Breger JA, Beauvais A, Liu H et al. Loss of cell wall alpha(1-3) glucan affects Cryptococcus neoformans from ultrastructure to virulence. Mol Microbiol 2007;63:1385–1398 [CrossRef][PubMed]
    [Google Scholar]
  9. Rodrigues ML, Alvarez M, Fonseca FL, Casadevall A. Binding of the wheat germ lectin to Cryptococcus neoformans suggests an association of chitinlike structures with yeast budding and capsular glucuronoxylomannan. Eukaryot Cell 2008;7:602–609 [CrossRef][PubMed]
    [Google Scholar]
  10. Fonseca FL, Nimrichter L, Cordero RJ, Frases S, Rodrigues J et al. Role for chitin and chitooligomers in the capsular architecture of Cryptococcus neoformans. Eukaryot Cell 2009;8:1543–1553 [CrossRef][PubMed]
    [Google Scholar]
  11. Nosanchuk JD, Casadevall A. Budding of melanized Cryptococcus neoformans in the presence or absence of L-dopa. Microbiology 2003;149:1945–1951 [CrossRef][PubMed]
    [Google Scholar]
  12. Eisenman HC, Frases S, Nicola AM, Rodrigues ML, Casadevall A. Vesicle-associated melanization in Cryptococcus neoformans. Microbiology 2009;155:3860–3867 [CrossRef][PubMed]
    [Google Scholar]
  13. Free SJ. Fungal cell wall organization and biosynthesis. Adv Genet 2013;81:33–82 [CrossRef][PubMed]
    [Google Scholar]
  14. Doering TL. How sweet it is! Cell wall biogenesis and polysaccharide capsule formation in Cryptococcus neoformans. Annu Rev Microbiol 2009;63:223–247 [CrossRef][PubMed]
    [Google Scholar]
  15. Lenardon MD, Whitton RK, Munro CA, Marshall D, Gow NA. Individual chitin synthase enzymes synthesize microfibrils of differing structure at specific locations in the Candida albicans cell wall. Mol Microbiol 2007;66:1164–1173 [CrossRef][PubMed]
    [Google Scholar]
  16. Araki Y, Ito E. A pathway of chitosan formation in Mucor rouxii. Enzymatic deacetylation of chitin. Eur J Biochem 1975;55:71–78 [CrossRef][PubMed]
    [Google Scholar]
  17. Kafetzopoulos D, Martinou A, Bouriotis V. Bioconversion of chitin to chitosan: purification and characterization of chitin deacetylase from Mucor rouxii. Proc Natl Acad Sci USA 1993;90:2564–2568 [CrossRef][PubMed]
    [Google Scholar]
  18. 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 [CrossRef][PubMed]
    [Google Scholar]
  19. 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 [CrossRef][PubMed]
    [Google Scholar]
  20. Casadevall A, Cordero RJB, Bryan R, Nosanchuk J, Dadachova E. Melanin, radiation, and energy transduction in fungi. Microbiol Spectr 2017;5:1–6 [CrossRef][PubMed]
    [Google Scholar]
  21. Nosanchuk JD, Casadevall A. Impact of melanin on microbial virulence and clinical resistance to antimicrobial compounds. Antimicrob Agents Chemother 2006;50:3519–3528 [CrossRef][PubMed]
    [Google Scholar]
  22. Casadevall A, Rosas AL, Nosanchuk JD. Melanin and virulence in Cryptococcus neoformans. Curr Opin Microbiol 2000;3:354–358 [CrossRef][PubMed]
    [Google Scholar]
  23. Eisenman HC, Casadevall A. Synthesis and assembly of fungal melanin. Appl Microbiol Biotechnol 2012;93:931–940 [CrossRef][PubMed]
    [Google Scholar]
  24. Chatterjee S, Prados-Rosales R, Itin B, Casadevall A, Stark RE. Solid-state NMR reveals the carbon-based molecular architecture of Cryptococcus neoformans fungal eumelanins in the cell wall. J Biol Chem 2015;290:13779–13790 [CrossRef][PubMed]
    [Google Scholar]
  25. Chatterjee S, Prados-Rosales R, Tan S, Itin B, Casadevall A et al. Demonstration of a common indole-based aromatic core in natural and synthetic eumelanins by solid-state NMR. Org Biomol Chem 2014;12:6730–6736 [CrossRef][PubMed]
    [Google Scholar]
  26. Chatterjee S, Prados-Rosales R, Frases S, Itin B, Casadevall A et al. Using solid-state NMR to monitor the molecular consequences of Cryptococcus neoformans melanization with different catecholamine precursors. Biochemistry 2012;51:6080–6088 [CrossRef][PubMed]
    [Google Scholar]
  27. 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 [CrossRef][PubMed]
    [Google Scholar]
  28. Tian S, Garcia-Rivera J, Yan B, Casadevall A, Stark RE. Unlocking the molecular structure of fungal melanin using 13C biosynthetic labeling and solid-state NMR. Biochemistry 2003;42:8105–8109 [CrossRef][PubMed]
    [Google Scholar]
  29. 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 [CrossRef][PubMed]
    [Google Scholar]
  30. Bakulin AV, Veleshko IE, Rumyantseva EV, Levov AN, Burmistrova LA et al. Production of chitin—melanin complexes from Apis mellifera and study of the possibility of using them as radionuclide sorbents. Russ Agric Sci 2011;37:423–426 [CrossRef]
    [Google Scholar]
  31. Hwang DS, Masic A, Prajatelistia E, Iordachescu M, Waite JH. Marine hydroid perisarc: a chitin- and melanin-reinforced composite with DOPA-iron(III) complexes. Acta Biomater 2013;9:8110–8117 [CrossRef][PubMed]
    [Google Scholar]
  32. Stavenga DG, Leertouwer HL, Hariyama T, de Raedt HA, Wilts BD. Sexual dichromatism of the damselfly Calopteryx japonica caused by a melanin-chitin multilayer in the male wing veins. PLoS One 2012;7:e49743 [CrossRef][PubMed]
    [Google Scholar]
  33. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 2012;9:676–682 [CrossRef][PubMed]
    [Google Scholar]
  34. Smith RL, Gilkerson E. Quantitation of glycosaminoglycan hexosamine using 3-methyl-2-benzothiazolone hydrazone hydrochloride. Anal Biochem 1979;98:478–480 [CrossRef][PubMed]
    [Google Scholar]
  35. Ruiz-Herrera J, Mormeneo S, Vanaclocha P, Font-de-Mora J, Iranzo M et al. Structural organization of the components of the cell wall from Candida albicans. Microbiology 1994;140:1513–1523 [CrossRef][PubMed]
    [Google Scholar]
  36. García-Rodas R, Trevijano-Contador N, Román E, Janbon G, Moyrand F et al. Role of Cln1 during melanization of Cryptococcus neoformans. Front Microbiol 2015;6:798 [CrossRef][PubMed]
    [Google Scholar]
  37. Hu G, Cheng PY, Sham A, Perfect JR, Kronstad JW. Metabolic adaptation in Cryptococcus neoformans during early murine pulmonary infection. Mol Microbiol 2008;69:1456–1475 [CrossRef][PubMed]
    [Google Scholar]
  38. Fung BM, Khitrin AK, Ermolaev K. An improved broadband decoupling sequence for liquid crystals and solids. J Magn Reson 2000;142:97–101 [CrossRef][PubMed]
    [Google Scholar]
  39. Ost KS, Esher SK, Leopold Wager CM, Walker L, Wagener J et al. Rim pathway-mediated alterations in the fungal cell wall influence immune recognition and inflammation. MBio 2017;8:e02290-16 [CrossRef][PubMed]
    [Google Scholar]
  40. François JM. A simple method for quantitative determination of polysaccharides in fungal cell walls. Nat Protoc 2006;1:2995–3000 [CrossRef][PubMed]
    [Google Scholar]
  41. 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]
  42. Watson HR, Apperley DC, Dixon DP, Edwards R, Hodgson DR. An efficient method for 15N-labeling of chitin in fungi. Biomacromolecules 2009;10:793–797 [CrossRef][PubMed]
    [Google Scholar]
  43. Bartnicki-Garcia S, Lippman E. Fungal morphogenesis: cell wall construction in Mucor rouxii. Science 1969;165:302–304 [CrossRef][PubMed]
    [Google Scholar]
  44. James PG, Cherniak R, Jones RG, Stortz CA, Reiss E. Cell-wall glucans of Cryptococcus neoformans CAP 67. Carbohydr Res 1990;198:23–38 [CrossRef][PubMed]
    [Google Scholar]
  45. Sakaguchi N, Baba T, Fukuzawa M, Ohno S. Ultrastructural study of Cryptococcus neoformans by quick-freezing and deep-etching method. Mycopathologia 1993;121:133–141 [CrossRef][PubMed]
    [Google Scholar]
  46. Chaffin WL, López-Ribot JL, Casanova M, Gozalbo D, Martínez JP. Cell wall and secreted proteins of Candida albicans: identification, function, and expression. Microbiol Mol Biol Rev 1998;62:130–180[PubMed]
    [Google Scholar]
  47. Eisenman HC, Chow SK, Tsé KK, Mcclelland EE, Casadevall A. The effect of L-DOPA on Cryptococcus neoformans growth and gene expression. Virulence 2011;2:329–336 [CrossRef][PubMed]
    [Google Scholar]
  48. 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 [CrossRef][PubMed]
    [Google Scholar]
  49. Williamson PR. Biochemical and molecular characterization of the diphenol oxidase of Cryptococcus neoformans: identification as a laccase. J Bacteriol 1994;176:656–664 [CrossRef][PubMed]
    [Google Scholar]
  50. Ene IV, Adya AK, Wehmeier S, Brand AC, Maccallum DM et al. Host carbon sources modulate cell wall architecture, drug resistance and virulence in a fungal pathogen. Cell Microbiol 2012;14:1319–1335 [CrossRef][PubMed]
    [Google Scholar]
  51. Ene IV, Walker LA, Schiavone M, Lee KK, Martin-Yken H et al. Cell wall remodeling enzymes modulate fungal cell wall elasticity and osmotic stress resistance. MBio 2015;6:e00986 [CrossRef][PubMed]
    [Google Scholar]
  52. Du H, Guan G, Li X, Gulati M, Tao L et al. N-acetylglucosamine-induced cell death in Candida albicans and its implications for adaptive mechanisms of nutrient sensing in yeasts. MBio 2015;6:e01376-15 [CrossRef][PubMed]
    [Google Scholar]
  53. Nadal M, Sawers R, Naseem S, Bassin B, Kulicke C et al. An N-acetylglucosamine transporter required for arbuscular mycorrhizal symbioses in rice and maize. Nat Plants 2017;3:17073 [CrossRef][PubMed]
    [Google Scholar]
  54. Zaragoza O, Chrisman CJ, Castelli MV, Frases S, Cuenca-Estrella M et al. Capsule enlargement in Cryptococcus neoformans confers resistance to oxidative stress suggesting a mechanism for intracellular survival. Cell Microbiol 2008;10:2043–2057 [CrossRef][PubMed]
    [Google Scholar]
  55. Córdoba S, Afeltra J, Vitale RG. Evaluation of the in vitro activity of amphotericin B by time-kill curve methodology against large and small capsulate C. neoformans isolates. Diagn Microbiol Infect Dis 2011;71:260–262 [CrossRef][PubMed]
    [Google Scholar]
  56. Bouklas T, Fries BC. Aging as an emergent factor that contributes to phenotypic variation in Cryptococcus neoformans. Fungal Genet Biol 2015;78:59–64 [CrossRef][PubMed]
    [Google Scholar]
  57. 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]
  58. Casadevall A, Steenbergen JN, Nosanchuk JD. 'Ready made' virulence and 'dual use' virulence factors in pathogenic environmental fungi-the Cryptococcus neoformans paradigm. Curr Opin Microbiol 2003;6:332–337 [CrossRef][PubMed]
    [Google Scholar]
  59. Gerstein AC, Nielsen K. It's not all about us: evolution and maintenance of Cryptococcus virulence requires selection outside the human host. Yeast 2017;34:143–154 [CrossRef][PubMed]
    [Google Scholar]
  60. Younes I, Rinaudo M. Chitin and chitosan preparation from marine sources. Structure, properties and applications. Mar Drugs 2015;13:1133–1174 [CrossRef][PubMed]
    [Google Scholar]
  61. Konopka JB. N-acetylglucosamine (GlcNAc) functions in cell signaling. Scientifica 2012;2012:1–15 [CrossRef]
    [Google Scholar]
  62. Sudbery PE. Growth of Candida albicans hyphae. Nat Rev Microbiol 2011;9:737–748 [CrossRef][PubMed]
    [Google Scholar]
  63. Yamada-Okabe T, Sakamori Y, Mio T, Yamada-Okabe H. Identification and characterization of the genes for N-acetylglucosamine kinase and N-acetylglucosamine-phosphate deacetylase in the pathogenic fungus Candida albicans. Eur J Biochem 2001;268:2498–2505 [CrossRef][PubMed]
    [Google Scholar]
  64. Cannon RD, Niimi K, Jenkinson HF, Shepherd MG. Molecular cloning and expression of the Candida albicans beta-N-acetylglucosaminidase (HEX1) gene. J Bacteriol 1994;176:2640–2647 [CrossRef][PubMed]
    [Google Scholar]
  65. Baker LG, Specht CA, Lodge JK. Chitinases are essential for sexual development but not vegetative growth in Cryptococcus neoformans. Eukaryot Cell 2009;8:1692–1705 [CrossRef][PubMed]
    [Google Scholar]
  66. Cleare W, Casadevall A. Scanning electron microscopy of encapsulated and non-encapsulated Cryptococcus neoformans and the effect of glucose on capsular polysaccharide release. Med Mycol 1999;37:235–243[PubMed]
    [Google Scholar]
  67. Revankar SG, Fu J, Rinaldi MG, Kelly SL, Kelly DE et al. Cloning and characterization of the lanosterol 14alpha-demethylase (ERG11) gene in Cryptococcus neoformans. Biochem Biophys Res Commun 2004;324:719–728 [CrossRef][PubMed]
    [Google Scholar]
  68. Nosanchuk JD, Stark RE, Casadevall A. Fungal melanin: what do we know about structure?. Front Microbiol 2015;6:1463 [CrossRef][PubMed]
    [Google Scholar]
  69. Feldmesser M, Kress Y, Casadevall A. Dynamic changes in the morphology of Cryptococcus neoformans during murine pulmonary infection. Microbiology 2001;147:2355–2365 [CrossRef][PubMed]
    [Google Scholar]
  70. 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 [CrossRef][PubMed]
    [Google Scholar]
  71. Merzendorfer H, Zimoch L. Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases. J Exp Biol 2003;206:4393–4412 [CrossRef][PubMed]
    [Google Scholar]
  72. Ahmed TA, Aljaeid BM. Preparation, characterization, and potential application of chitosan, chitosan derivatives, and chitosan metal nanoparticles in pharmaceutical drug delivery. Drug Des Devel Ther 2016;10:483–507 [CrossRef][PubMed]
    [Google Scholar]
  73. Quemeneur F, Rinaudo M, Pépin-Donat B. Influence of molecular weight and pH on adsorption of chitosan at the surface of large and giant vesicles. Biomacromolecules 2008;9:396–402 [CrossRef][PubMed]
    [Google Scholar]
  74. Nosanchuk JD, Casadevall A. Cellular charge of Cryptococcus neoformans: contributions from the capsular polysaccharide, melanin, and monoclonal antibody binding. Infect Immun 1997;65:1836–1841[PubMed]
    [Google Scholar]
  75. Zhu X, Williamson PR. Role of laccase in the biology and virulence of Cryptococcus neoformans. FEMS Yeast Res 2004;5:1–10 [CrossRef][PubMed]
    [Google Scholar]
  76. Zhang Y, Thomas Y, Kim E, Payne GF. pH- and voltage-responsive chitosan hydrogel through covalent cross-linking with catechol. J Phys Chem B 2012;116:1579–1585 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000552
Loading
/content/journal/micro/10.1099/mic.0.000552
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error