| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Departments of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA
2 Departments of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY, USA
Correspondence
Joshua D. Nosanchuk
nosanchu{at}aecom.yu.edu
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
, 1963) and was associated with virulence in the 1980s (Kwon-Chung et al., 1982; Rhodes et al., 1982). Melanins are rigid, acid-resistant polymers of uncertain structure that are found in all biological kingdoms (Hill, 1992). The synthesis of melanin by C. neoformans occurs only in the presence of phenolic compounds, such as L-3,4-dihydroxyphenylalanine (L-dopa), and is catalysed by a laccase (Polacheck et al., 1982; Williamson, 1994). The C. neoformans laccase has recently been shown to be tightly associated with the cell wall by a hydrolysable bond (Zhu et al., 2001). When the fungus grows in the presence of phenolic compounds, melanin is deposited in the cell wall (Wang et al., 1995) and comprises approximately 15 % of the dry weight of late-stationary-phase melanized C. neoformans (Wang & Casadevall, 1996).
C. neoformans is melanized in the environment (Nosanchuk et al., 1999b) and during human infection (Nosanchuk et al., 2000). In the environment melanin is thought to protect the fungus against extremes of temperature (Rosas & Casadevall, 1997), ultraviolet light (Wang & Casadevall, 1994a), heavy metals (Garcia-Rivera & Casadevall, 2001) and amoeboid predators (Steenbergen et al., 2001). During infection melanin appears to function in virulence by protecting fungal cells against microbicidal oxidants (Wang & Casadevall, 1994b) and peptides (Doering et al., 1999) produced by immune effector cells. Furthermore, melanin may interfere with the development of effective cell-mediated responses (Huffnagle et al., 1995) and could affect the activation of complement (Rosas et al., 2002) in infected hosts. Melanin can also contribute to the difficulty associated with the treatment of C. neoformans infections with antifungal drugs, since melanin can reduce the susceptibility of pigmented cells to amphotericin B and caspofungin (Van Duin et al., 2002). Given that melanin appears to increase fungal fitness in vivo, it is a potential target for drug discovery. In this regard, treatment of infected mice with glyphosate, an inhibitor of melanization, was associated with a beneficial therapeutic effect (Nosanchuk et al., 2001).
Melanization of C. neoformans results in the deposition of the polymer in the cell wall. Treatment of melanized cells with enzymes, detergents and hot acid results in the recovery of melanin ‘ghosts' that retain the size and shape of the original fungal cells (Rosas et al., 2000a). Although the molecular structure of melanin is unknown, it is presumed to be composed of a lattice of interlocking, covalently bonded aromatic and aliphatic structures (Bell & Wheeler, 1986; Wheeler & Bell, 1988). Hence, melanin is a dense, rigid and amorphous material with significant material strength. In fact, melanization is believed to provide great rigidity to hyphal cells, which may aid in the penetration of plants by melanotic pathogenic fungi (Howard et al., 1991; Money, 1997; Money et al., 1998). Melanin synthesis may also provide a biomechanical advantage for invasive hyphal growth of the human-pathogenic fungus Exophiala (Wangiella) dermatitidis (Brush & Money, 1999; Dixon et al., 1989, 1991).
Since melanin is located in the cell wall of pigmented C. neoformans, the fungus must bud through this dense polymer to replicate. Given the structural toughness of this pigment, budding in the presence of melanin is likely to be a complex process, requiring both degradation and synthesis of the polymer concurrently in the mother and daughter cells. Despite the biological importance of melanin and the widespread occurrence of melanin in the mycota, we are not aware of any published information as to the effect of budding on melanized cells. In this study we investigated the effect of budding on melanized and non-melanized C. neoformans cells in the presence and absence of substrate for melanin synthesis.
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). The cells from the 10-day-old cultures were collected by centrifugation at 320 g for 10 min, washed 3 times in PBS and approximately 100 000 organisms were inoculated into 100 ml fresh minimal medium with or without L-dopa. Samples from the subcultures were removed at different intervals and cell counts were determined by haemocytometer.
Immunofluorescence.
Cells from 10-day-old melanized and non-melanized C. neoformans cultures transferred to fresh medium with or without 1·0 mM L-dopa were collected after 36 h growth in fresh medium and washed. For immunofluorescence analyses, suspensions of C. neoformans cells were air-dried on poly-L-lysine slides (Sigma), then blocked for non-specific binding with Superblock (Pierce) for 1 h at 37 °C. The slides were then incubated with 10 µg melanin-binding mAb 6D2 ml-1 overnight at 4 °C. mAb 6D2 (µ
) was generated against melanin derived from C. neoformans and also binds other types of melanins, but does not bind Candida albicans, Saccharomyces cerevisiae or a laccase-deficient mutant of C. neoformans (Rosas et al., 2000b). After washing, the sections were incubated with a 1 : 1000 dilution of fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgM (Southern Biotechnologies Associates) for 1 h at 37 °C. The sections were washed, coverslips were mounted using a 50 % glycerol, 50 % PBS and 0·1 M N-propyl gallate solution, and viewed with an Olympus AX70 microscope equipped with a FITC filter. Negative controls consisted of cells incubated with mAb 5C11 (µ), which binds mycobacterial lipoarabinomannan (Glatman-Freedman et al., 1996), as the primary antibody or with FITC-labelled antibody alone. The non-melanized cells that were transferred to fresh medium without L-dopa also served as negative controls.
Isolation of melanin ‘ghosts' from C. neoformans.
Treatment of melanized C. neoformans cells with enzymes, denaturant and hot acid results in the isolation of purified melanin in the shape and size of the parental melanized cryptococcal cell, and these particles are referred to as melanin ‘ghosts' (Wang & Casadevall, 1996). Briefly, C. neoformans from the subcultures of cells grown for 10 days and transferred to fresh medium with or without L-dopa for 36 h were collected by centrifugation at 2010 g for 30 min, washed with PBS and suspended in 1·0 M sorbitol/0·1 M sodium citrate (pH 5·5). Cell-wall-lysing enzymes (from Trichoderma harzianum; Sigma) were added at 10 mg ml-1 and the suspensions were incubated at 30 °C overnight. The resulting protoplasts were collected by centrifugation, washed with PBS and treated with 1 mg proteinase K ml-1 (Roche Laboratories) made up in a reaction buffer (10 mM Tris, 1 mM CaCl2 and 0·5 % SDS; pH 7·8) at 37 °C overnight. The debris was collected, washed with PBS and then boiled in 6 M HCl for 1 h. If particles remained, they were collected, washed in PBS and dialysed extensively against distilled water. This procedure has been shown to solubilize non-melanized C. neoformans, Paracoccidioides brasiliensis, S. cerevisiae and Candida albicans yeast cells (Gómez et al., 2001; Rosas et al., 2000b).
Scanning electron microscopy (SEM).
The particles isolated from the treated cells were studied by SEM as described previously (Nosanchuk et al., 1999a). Samples were incubated in 2·5 % gluteraldehyde in 0·1 M cacodylate for 1 h at room temperature, applied to poly-L-lysine-coated coverslips and serially dehydrated in alcohol. The samples were dried (Samdri-790; Tousimis), coated with gold-palladium (Desk-1; Denton Vacuum) and viewed using a JEOL JAM-6400 electron microscope.
Transmission electron microscopy (TEM).
Samples of melanized and non-melanized C. neoformans were high-pressure-frozen using a Leica EMpact High Pressure Freezer. Frozen samples were transferred to a Leica EM AFS Freeze Substitution Unit and freeze-substituted in 1 % osmium tetroxide in acetone. They were brought from -90 °C to room temperature over 2–3 days, rinsed in acetone and embedded in Spurrs epoxy resin (Polysciences). Samples of ‘ghosts' were fixed in 2 % gluteraldehyde in 0·1 M cacodylate at room temperature for 2 h followed by an overnight incubation in 4 % formaldehyde/1 % gluteraldehyde/0·1 % PBS. The samples were subjected to a 1·5 h post fixation in 2 % osmium, serially dehydrated in ethanol and embedded in Spurrs epoxy resin. Ultrathin sections of 70–80 nm were cut on a Reichart Ultracut UCT and stained with uranyl acetate followed by lead citrate. Samples were viewed on a JEOL 1200EX transmission electron microscope at 80 kV.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
, 1997). Similar results were obtained with C. neoformans strains 3501 and CAP67. Hence, the melanized state did not slow the rate of growth of C. neoformans.
Immunofluorescence
Melanin-binding mAb only labelled cells from C. neoformans cultures grown for 10 days with L-dopa. All of the melanized cells transferred to fresh medium with L-dopa were labelled by the melanin-binding mAb and the buds on replicating cells were similarly labelled (Fig. 1a). In contrast, approximately 30 % of cells from the cultures without substrate initiated with previously melanized cells were labelled by the melanin-binding mAb. Additionally, only the parental portion of budding cells was labelled (Fig. 1b). Similar findings were seen with each of the C. neoformans strains examined, but the immunofluorescent staining by the melanin-binding mAb was superior with CAP67, which lacks a polysaccharide capsule that impedes antibody from reaching the cell wall.
|
) (Wang & Casadevall, 1996). As expected, no ‘ghosts' were obtained from cells collected from samples grown initially without substrate, whether or not they were transferred to medium with L-dopa for 36 h. Non-melanized, stationary-phase cells are apparently unable to form a ‘shell’ of polymerized melanin over this period of time. The ‘ghosts' isolated from melanized cells transferred to medium with L-dopa were a variety of sizes (diam. 4·7±1·5 µm; range 1·5–7 µm), consistent with originating from a mixed population of melanized cells composed of older parental and recently budded cells. Budding cells demonstrated a connection of melanin between the parental and daughter cells (Fig. 2a). When melanized cells were grown in the absence of phenolic compounds, no budding-cell ‘ghosts' were observed and the cells were more uniform in size (diam. 5·3±0·6 µm; range 4·5–7 µm), consistent with the solubilization of all but the melanized parental cells. However, in these conditions the majority of ‘ghosts' had defects, or ‘holes' (Fig. 2b). These defects presumably represented locations of cell budding on the mother cell where non-melanized daughter cells were attached prior to being solubilized by treatment with enzymes and chemicals.
|
a, b). In contrast, imaging of ‘ghosts' generated from melanized cells showed defects in cross section at sites of apparent budding that are likely to correspond to the defects in the melanin layer observed by SEM (Fig. 3c). Hence, cell division for a melanized cell is accomplished by the formation of a break in the melanin layer where budding occurs.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
) and in pigeon excreta (Nosanchuk et al., 1999b). Relatively little is known about the mechanisms of budding in C. neoformans (Kopecka et al., 2001) and there is no information available regarding budding in the presence of melanin or about the production of melanin during budding. Given that melanized cells grow and replicate, we postulated that rearrangement of melanin must occur despite the fact that this polymer is rigid, dense and tenacious. Although no specific melanin-degrading enzymes have been described, such enzymes must exist given the abundance of this material in nature. Degradative processes have been identified, for the related polymer lignin and ligninases of Phanerochaete chrysosporium have been shown to destroy fungal melanins (Butler & Day, 1998). Interestingly, the white rot fungus, Pycnoporus cinnabarinus, produces a metabolite, 3-hydroxyanthranilate, that mediates the oxidation of non-phenolic substrates by laccase that results in the depolymerization of lignin (Eggert et al., 1996b) and laccase is essential for the lignolytic ability of the fungus (Eggert et al., 1996a, 1997). Decolourization of melanin by the fungi Bjerkandera adusta, Galactomyces geotrichum, Trametes hirsuta and Trametes versicolor is a phenomenon consistent with the destruction of the pigment, which provides further evidence for the presence of melanin-degrading enzymes (Ratto et al., 2001). Additionally, Aspergillus fumigatus, an important human-pathogenic fungus that synthesizes melanin (Jahn et al., 1997; Tsai et al., 2001), can utilize environmental melanins as a sole carbon source for growth, indicating that the fungus is capable of degrading melanin (Luther & Lipke, 1980). Hence, there is considerable evidence that melanin-degrading enzymic mechanisms exist. Our results show that replication of pigmented C. neoformans cells is accomplished by the formation of a ‘hole’ in the melanin layer at the site of budding. The occurrence of a localized defect in conjunction with cell replication is consistent with an enzymic process linked to the cell cycle that results in the digestion of a section of the melanin layer. These findings establish that although C. neoformans can apparently rearrange melanin, buds do not take significant amounts of melanin from parental cells. After budding, the parental cell must form new melanin at the bud scar. The lack of phenolic substrate does not affect the growth of either melanized or non-melanized yeast cells.
The results obtained can be interpreted to synthesize a coherent model for the replication of melanized cells in the presence or absence of substrate for melanin (Fig. 4). Bud formation in melanized cells must initially be accompanied by a breakdown of cell-wall-associated melanin at the site of a newly forming bud. In the mother cell, we postulate that enzymic processes digest the melanin and then reform the polymer at the bud scar upon completion of budding if substrate for melanin synthesis is available. Concurrently, the bud synthesizes melanin de novo and the daughter cell is melanized. When substrate is not available, a defect occurs in the mother cell's melanin layer and the daughter cell is not melanized.
|
). The capsular polysaccharide of C. neoformans is thinner over budding cells (Cassone et al., 1974) and qualitatively different from the parental polysaccharide (Feldmesser et al., 2000). In fact, the capsule of buds results from new synthesis rather than from parasitism of capsule present on the parent cell (Pierini & Doering, 2001). The findings of de novo synthesis of melanin and capsule by buds of C. neoformans is consistent with prior data demonstrating localized synthesis of other cell wall constituents in buds of Candida albicans (Osumi, 1998) and S. cerevisiae (Cid et al., 2002; Drees et al., 2001; Sekiya-Kawasaki et al., 2002).
The presence of melanin can significantly protect C. neoformans from the antifungal compounds amphotericin B and caspofungin by binding the antifungals, which reduces their availability and efficacy (Van Duin et al., 2002). Disruption of melanin in the cell wall during budding may provide an avenue for drug entry into C. neoformans. Glyphosate, an inhibitor of the shikimate acid pathway, inhibits melanization of C. neoformans in vitro and prolongs the survival of mice with cryptococcosis (Nosanchuk et al., 2001). This demonstrates that inhibition of melanin production in wild-type C. neoformans has important biological significance. Similarly, mAbs to melanin and melanin-binding peptides inhibit the growth of melanized cells and melanin-binding mAbs prolong survival in mice infected with C. neoformans (Rosas et al., 2001). The mechanism for inhibition of cell growth by melanin-binding peptides and mAbs is unknown, but may be due to alterations in the properties of the melanin in the cell wall or by blocking of pores in melanin that permit the entry of essential nutrients for C. neoformans. The effects of these compounds on budding were not directly assessed; however, budding represents a period of heightened cellular activity during which reagents targeting melanin production or other synthetic processes may be most affected.
Our observations imply a very complex regulation of melanin metabolism and linkage of melanin degradation and synthesis to the cell cycle. Melanin is one of several virulence factors linked to cAMP-associated signalling pathways (Alspaugh et al., 1997, 1998). Disruption of a gene encoding a cAMP-dependent protein kinase results in mutants that are avirulent, as they do not produce melanin or a polysaccharide capsule (D'Souza et al., 2001). Melanin synthesis and pathogenicity of C. neoformans are significantly impaired by the down-regulation of the gene responsible for an inositol-phosphoryl ceramide synthase (Luberto et al., 2001). Avirulent mutant strains that have reduced melanin synthesis, capsule production, urease expression and growth at 37 °C have been generated by disruption of a gene encoding a vesicular proton pump (Erickson et al., 2001). These findings show that the regulation of melanin synthesis is associated with several important virulence determinants and suggest that targeting enzymes involved in melanin production and rearrangement may have a broad impact on C. neoformans virulence.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Alspaugh, J. A., Perfect, J. R. & Heitman, J. (1998). Signal transduction pathways regulating differentiation and pathogenicity of Cryptococcus neoformans. Fungal Genet Biol 25, 1–14.[CrossRef][Medline]
Bell, A. A. & Wheeler, M. H. (1986). Biosynthesis and functions of fungal melanins. Annu Rev Phytopathol 24, 411–451.[CrossRef]
Brush, L. & Money, N. P. (1999). Invasive hyphal growth in Wangiella dermatitidis is induced by stab inoculation and shows dependence upon melanin biosynthesis. Fungal Genet Biol 28, 190–200.[CrossRef][Medline]
Butler, M. J. & Day, A. W. (1998). Ligninases are melaninases. Int J Plant Sci 159, 989–995.[CrossRef]
Cassone, A., Simonetti, N. & Strippoli, V. (1974). Wall structure and bud formation on Cryptococcus neoformans. Arch Microbiol 95, 205–212.[CrossRef]
Chen, L. C., Blank, E. S. & Casadevall, A. (1996). Extracellular proteinase activity of Cryptococcus neoformans. Clin Diagn Lab Immunol 3, 570–574.[Abstract]
Chen, L. C., Pirofski, L. A. & Casadevall, A. (1997). Extracellular proteins of Cryptococcus neoformans and host antibody response. Infect Immun 65, 2599–2605.[Abstract]
Cid, V. J., Jiménez, J., Molina, M., Sánchez, M., Nombela, C. & Thorner, J. W. (2002). Orchestrating the cell cycle in yeast: sequential localization of key mitotic regulators at the spindle pole and the bud neck. Microbiology 148, 2647–2659.
Dixon, D. M., Polak, A. & Conner, G. W. (1989). Mel- mutants of Wangiella dermatitidis in mice: evaluation of multiple mouse and fungal strains. J Med Vet Mycol 27, 335–341.[Medline]
Dixon, D. M., Szaniszlo, P. J. & Polak, A. (1991). Dihydroxynaphthalene (DHN) melanin and its relationship with virulence in the early stages of phaeohyphomycosis. In The Fungal Spore and Disease Initiation in Plants and Animals, pp. 297–318. Edited by G. Cole & H. Hoch. New York: Plenum.
Doering, T. L., Nosanchuk, J. D., Roberts, W. K. & Casadevall, A. (1999). Melanin as a potential cryptococcal defence against microbicidal proteins. Med Mycol 37, 175–181.[CrossRef][Medline]
Drees, B. L., Sundin, B., Brazeau, E. & 19 other authors (2001). A protein interaction map for cell polarity development. J Cell Biol 154, 549–571.
D'Souza, C. A., Alspaugh, J. A., Yue, C., Harashima, T., Cox, G. M., Perfect, J. R. & Heitman, J. (2001). Cyclic AMP-dependent protein kinase controls virulence of the fungal pathogen Cryptococcus neoformans. Mol Cell Biol 21, 3179–3191.
Eggert, C., Temp, U. & Eriksson, K. E. (1996a). The ligninolytic system of the white rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase. Appl Environ Microbiol 62, 1151–1158.[Abstract]
Eggert, C., Temp, U., Dean, J. F. & Eriksson, K. E. (1996b). A fungal metabolite mediates degradation of non-phenolic lignin structures and synthetic lignin by laccase. FEBS Lett 391, 144–148.[CrossRef][Medline]
Eggert, C., Temp, U. & Eriksson, K. E. (1997). Laccase is essential for lignin degradation by the white-rot fungus Pycnoporus cinnabarinus. FEBS Lett 407, 89–92.[CrossRef][Medline]
Erickson, T., Liu, L., Gueyikian, A., Zhu, X., Gibbons, J. & Williamson, P. R. (2001). Multiple virulence factors of Cryptococcus neoformans are dependent on VPH1. Mol Microbiol 42, 1121–1131.[CrossRef][Medline]
Feldmesser, M., Rivera, J., Kress, Y., Kozel, T. R. & Casadevall, A. (2000). Antibody interactions with the capsule of Cryptococcus neoformans. Infect Immun 68, 3642–3650.
Garcia-Rivera, J. & Casadevall, A. (2001). Melanization of Cryptococcus neoformans reduces its susceptibility to the antimicrobial effects of silver nitrate. Med Mycol 39, 353–357.[Medline]
Glatman-Freedman, A., Martin, J. M., Riska, P. F., Bloom, B. R. & Casadevall, A. (1996). Monoclonal antibodies to surface antigens of Mycobacterium tuberculosis and their use in a modified enzyme-linked immunosorbent spot assay for detection of mycobacteria. J Clin Microbiol 34, 2795–2802.[Abstract]
Gómez, B. L., Nosanchuk, J. D., Diez, S., Youngchim, S., Aisen, P., Cano, L. E., Restrepo, A., Casadevall, A. & Hamilton, A. J. (2001). Detection of melanin-like pigments in the dimorphic fungal pathogen Paracoccidioides brasiliensis in vitro and during infection. Infect Immun 69, 5760–5767.
Hill, Z. H. (1992). The function of melanin or six blind people examine an elephant. BioEssays 14, 49–56.[CrossRef][Medline]
Howard, R. J., Ferrari, M. A., Roach, D. H. & Money, N. P. (1991). Penetration of hard substrates by a fungus employing enormous turgor pressures. Proc Natl Acad Sci U S A 88, 11281–11284.
Huffnagle, G. B., Chen, G.-H., Curtis, J. L., McDonald, R. A., Strieter, R. M. & Toews, G. B. (1995). Down-regulation of the afferent phase of T cell-mediated pulmonary inflammation and immunity by a high melanin-producing strain of Cryptococcus neoformans. J Immunol 155, 3507–3516.[Abstract]
Jahn, B., Koch, A., Schmidt, A., Wanner, G., Gehringer, H., Bhakdi, S. & Brakhage, A. A. (1997). Isolation and characterization of a pigmentless-conidium mutant of Aspergillus fumigatus with altered conidial surface and reduced virulence. Infect Immun 65, 5110–5117.[Abstract]
Kopecka, M., Gabriel, M., Takeo, K., Yamaguchi, M., Svoboda, A., Ohkusu, M., Hata, K. & Yoshida, S. (2001). Microtubules and actin cytoskeleton in Cryptococcus neoformans compared with ascomycetous budding and fission yeasts. Eur J Cell Biol 80, 303–311.[CrossRef][Medline]
Kwon-Chung, K. J., Polacheck, I. & Popkin, T. J. (1982). Melanin-lacking mutants of Cryptococcus neoformans and their virulence for mice. J Bacteriol 150, 1414–1421.
Luberto, C., Toffaletti, D. L., Wills, E. A., Tucker, S. C., Casadevall, A., Perfect, J. R., Hannun, Y. A. & Del Poeta, M. M. (2001). Roles for inositol-phosphoryl ceramide synthase 1 (IPC1) in pathogenesis of C. neoformans. Genes Dev 15, 201–212.
Luther, J. P. & Lipke, H. (1980). Degradation of melanin by Aspergillus fumigatus. Appl Environ Microbiol 40, 145–155.
McFadden, D. C. & Casadevall, A. (2001). Capsule and melanin synthesis in Cryptococcus neoformans. Med Mycol 39, 19–30.
Money, N. P. (1997). Mechanism linking cellular pigmentation and pathogenicity in rice blast disease. Fungal Genet Biol 22, 151–152.[CrossRef][Medline]
Money, N. P., Caesar-TonThat, T. C., Frederick, B. & Henson, J. M. (1998). Melanin synthesis is associated with changes in hyphopodial turgor, permeability, and wall rigidity in Gaeumannomyces graminis var. graminis. Fungal Genet Biol 24, 240–251.[CrossRef][Medline]
Nosanchuk, J. D., Valadon, P., Feldmesser, M. & Casadevall, A. (1999a). Melanization of Cryptococcus neoformans in murine infection. Mol Cell Biol 19, 745–750.
Nosanchuk, J. D., Rudolph, J., Rosas, A. L. & Casadevall, A. (1999b). Evidence that Cryptococcus neoformans is melanized in pigeon excreta: implications for pathogenesis. Infect Immun 67, 5477–5479.
Nosanchuk, J. D., Rosas, A. L., Lee, S. C. & Casadevall, A. (2000). Melanisation of Cryptococcus neoformans in human brain tissue. Lancet 355, 2049–2050.[CrossRef][Medline]
Nosanchuk, J. D., Ovalle, R. & Casadevall, A. (2001). Glyphosate inhibits melanization of Cryptococcus neoformans and prolongs survival of mice after systemic infection. J Infect Dis 183, 1093–1099.[CrossRef][Medline]
Osumi, M. (1998). The ultrastructure of yeast: cell wall structure and formation. Micron 29, 207–233.
Pierini, L. M. & Doering, T. L. (2001). Spatial and temporal sequence of capsule construction in Cryptococcus neoformans. Mol Microbiol 41, 105–115.[CrossRef][Medline]
Polacheck, I., Hearing, V. J. & Kwon-Chung, K. J. (1982). Biochemical studies of phenoloxidase and utilization of catecholamines in Cryptococcus neoformans. J Bacteriol 150, 1212–1220.
Ratto, M., Chatani, M., Ritschkoff, A. C. & Viikari, L. (2001). Screening of micro-organisms for decolorization of melanins produced by bluestain fungi. Appl Microbiol Biotechnol 55, 210–213.[CrossRef][Medline]
Rhodes, J. C., Polacheck, I. & Kwon-Chung, K. J. (1982). Phenoloxidase activity and virulence in isogenic strains of Cryptococcus neoformans. Infect Immun 36, 1175–1184.
Rosas, A. L. & Casadevall, A. (1997). Melanization affects susceptibility of Cryptococcus neoformans to heat and cold. FEMS Microbiol Lett 153, 265–272.[Medline]
Rosas, A. L., Nosanchuk, J. D., Gomez, B. L., Edens, W. A., Henson, J. M. & Casadevall, A. (2000a). Isolation and serological analyses of fungal melanins. J Immunol Methods 244, 69–80.[CrossRef][Medline]
Rosas, A. L., Nosanchuk, J. D., Feldmesser, M., Cox, G. M., McDade, H. C. & Casadevall, A. (2000b). Synthesis of polymerized melanin by Cryptococcus neoformans in infected rodents. Infect Immun 68, 2845–2853.
Rosas, A. L., Nosanchuk, J. D. & Casadevall, A. (2001). Passive immunization with melanin-binding monoclonal antibodies prolongs survival of mice with lethal Cryptococcus neoformans infection. Infect Immun 69, 3410–3412.
Rosas, A. L., MacGill, R. S., Nosanchuk, J. D., Kozel, T. R. & Casadevall, A. (2002). Activation of the alternative complement pathway by fungal melanins. Clin Diagn Lab Immunol 9, 144–148.
Sekiya-Kawasaki, M., Abe, M., Saka, A., Watanabe, D., Kono, K., Minemura-Asakawa, M., Ishihara, S., Watanabe, T. & Ohya, Y. (2002). Dissection of upstream regulatory components of the Rho1p effector, 1,3-beta-glucan synthase, in Saccharomyces cerevisiae. Genetics 162, 663–676.
Staib, F. (1962). Cryptococcus neoformans and Guizotia abyssicnica (syn. G. oleifera D.C.). Z Hyg 148, 466–475.[CrossRef]
Staib, F. (1963). New concepts in the occurrence and identification of Cryptococcus neoformans. Mycopathol Mycol Appl 19, 143–145.
Steenbergen, J. N., Shuman, H. A. & Casadevall, A. (2001). Cryptococcus neoformans interactions with amoebae suggest an explanation for its virulence and intracellular pathogenic strategy in macrophages. Proc Natl Acad Sci U S A 11, 11.
Tsai, H. F., Fujii, I., Watanabe, A., Wheeler, M. H., Chang, Y. C., Yasuoka, Y., Ebizuka, Y. & Kwon-Chung, K. J. (2001). Pentaketide melanin biosynthesis in Aspergillus fumigatus requires chain-length shortening of a heptaketide precursor. J Biol Chem 276, 29292–29298.
Van Duin, D., Casadevall, A. & Nosanchuk, J. D. (2002). Melanization of Cryptococcus neoformans and Histoplasma capsulatum reduces their susceptibility to amphotericin B and caspofungin. Antimicrob Agents Chemother 46, 3394–3400.
Wang, Y. & Casadevall, A. (1994a). Decreased susceptibility of melanized Cryptococus neoformans to the fungicidal effects of ultraviolet light. Appl Environ Microbiol 60, 3864–3866.
Wang, Y. & Casadevall, A. (1994b). Susceptibility of melanized and nonmelanized Cryptococcus neoformans to nitrogen- and oxygen-derived oxidants. Infect Immun 62, 3004–3007.
Wang, Y. & Casadevall, A. (1996). Melanin, melanin ‘ghosts’, and melanin composition in Cryptococcus neoformans. Infect Immun 64, 2420–2424.[Abstract]
Wang, Y., Aisen, P. & Casadevall, A. (1995). Cryptococcus neoformans melanin and virulence: mechanism of action. Infect Immun 63, 3131–3136.[Abstract]
Wheeler, M. H. & Bell, A. A. (1988). Melanins and their importance in pathogenic fungi. Curr Topics Med Mycol 2, 338–387.[Medline]
Williamson, P. R. (1994). Biochemical and molecular characterization of the diphenol oxidase of Cryptococcus neoformans: identification as a laccase. J Bacteriol 176, 656–664.
Zhu, X., Gibbons, J., Garcia-Rivera, J., Casadevall, A. & Williamson, P. R. (2001). Laccase of Cryptococcus neoformans is a cell wall-associated virulence factor. Infect Immun 69, 5589–5596.
Received 7 March 2003;
revised 11 April 2003;
accepted 14 April 2003.
This article has been cited by other articles:
|
|
 |
|
  J. D. Nosanchuk and A. Casadevall Impact of Melanin on Microbial Virulence and Clinical Resistance to Antimicrobial Compounds Antimicrob. Agents Chemother., November 1, 2006; 50(11): 3519 - 3528. [Full Text] [PDF]
|
|
|
|
 |
|
 C. J. Clancy, M. H. Nguyen, R. Alandoerffer, S. Cheng, K. Iczkowski, M. Richardson, and J. R. Graybill Cryptococcus neoformans var. grubii isolates recovered from persons with AIDS demonstrate a wide range of virulence during murine meningoencephalitis that correlates with the expression of certain virulence factors. Microbiology, August 1, 2006; 152(Pt 8): 2247 - 2255. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Mandal, U. Banerjee, A. Casadevall, and J. D. Nosanchuk Dual Infections with Pigmented and Albino Strains of Cryptococcus neoformans in Patients with or without Human Immunodeficiency Virus Infection in India J. Clin. Microbiol., September 1, 2005; 43(9): 4766 - 4772. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Youngchim, R. Morris-Jones, R. J. Hay, and A. J. Hamilton Production of melanin by Aspergillus fumigatus J. Med. Microbiol., March 1, 2004; 53(3): 175 - 181. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
