Melanin Externalization in Candida albicans Depends on Cell Wall Chitin Structures

The fungal pathogen Candida albicans produces dark-pigmented melanin after 3 to 4 days of incubation in medium containing l-3,4-dihydroxyphenylalanine (l-DOPA) as a substrate. Expression profiling of C. albicans revealed very few genes significantly up- or downregulated by growth in l-DOPA. We were unable to determine a possible role for melanin in the virulence of C. albicans. However, we showed that melanin was externalized from the fungal cells in the form of electron-dense melanosomes that were free or often loosely bound to the cell wall exterior. Melanin production was boosted by the addition of N-acetylglucosamine to the medium, indicating a possible association between melanin production and chitin synthesis. Melanin externalization was blocked in a mutant specifically disrupted in the chitin synthase-encoding gene CHS2. Melanosomes remained within the outermost cell wall layers in chs3Δ and chs2Δ chs3Δ mutants but were fully externalized in chs8Δ and chs2Δ chs8Δ mutants. All the CHS mutants synthesized dark pigment at equivalent rates from mixed membrane fractions in vitro, suggesting it was the form of chitin structure produced by the enzymes, not the enzymes themselves, that was involved in the melanin externalization process. Mutants with single and double disruptions of the chitinase genes CHT2 and CHT3 and the chitin pathway regulator ECM33 also showed impaired melanin externalization. We hypothesize that the chitin product of Chs3 forms a scaffold essential for normal externalization of melanosomes, while the Chs8 chitin product, probably produced in cell walls in greater quantity in the absence of CHS2, impedes externalization.Candida albicans is a major opportunistic fungal human pathogen that causes a wide variety of infections (9, 68). In healthy individuals C. albicans resides as a commensal within the oral cavity and gastrointestinal and urogenital tracts. However, in immunocompromised hosts, C. albicans causes infections ranging in severity from mucocutaneous infections to life-threatening disseminated diseases (9, 68). Research into the pathogenicity of C. albicans has revealed a complex mix of putative virulence factors (7, 60), perhaps reflecting the fine balance this species strikes between commensal colonization and opportunistic invasion of the human host.Melanins are biological pigments, typically dark brown or black, formed by the oxidative polymerization of phenolic compounds. They are negatively charged hydrophobic molecules with high molecular weights and are insoluble in both aqueous and organic solvents. Their insolubility makes melanins difficult to study, and no definitive structure has yet been found for them; they probably represent an amorphous mixture of polymers (35). There are various types of melanin in nature, including eumelanin and phaeomelanin (76). Two principal types of melanin are found in the fungal kingdom. The majority are 1.8-dihydroxynapthalene (DNH) melanins synthesized from acetyl-coenzyme A (CoA) via the polyketide pathway (5). DNH melanins have been found in a wide range of opportunistic fungal pathogens of humans, including dark (dematiaceous) molds, such as Cladosporium, Fonsecaea, Phialophora, and Wangiella species, and as conidial pigments in Aspergillus fumigatus and Aspergillus niger (41, 80, 87, 88). However, several other fungal pathogens, including Blastomyces dermatitidis, Coccidioides posadasii, Cryptococcus neoformans, Histoplasma capsulatum, Paracoccidioides brasiliensis, and Sporothrix schenckii, produce eumelanin (3,4-dihydroxyphenylalanine [DOPA]-melanin) through the activity of a polyphenol oxidase (laccase) and require an exogenous o-diphenolic or p-diphenolic substrate, such as l-DOPA (16, 30, 63,–65, 67, 79).The production of melanin in humans and other mammals is a function of specialized cells called melanocytes. Particles of melanin polymers, sometimes, including more than one melanin type, are built up within membrane-bound organelles called melanosomes (76), and these are actively transported along microtubules to the tips of dendritic outgrowths of melanocytes, from where they are transferred to neighboring cells (32, 81). The mechanism of intercellular transfer of melanosomes has not yet been established, but the export process probably involves the fusion of cell and vesicular membranes rather than secretion of naked melanin (82). In pathogenic fungi, melanins are often reported to be associated with or “in” the cell wall (35, 36, 50, 72, 79). However, there is variation between species: the melanin may be located external to the wall, e.g., in P. brasiliensis (79); within the wall itself (reviewed in reference 42); or as a layer internal to the wall and external to the cell membrane, e.g., in C. neoformans (22, 45, 85). However, mutants of C. neoformans bearing disruptions of three CDA genes involved in the biosynthesis of cell wall chitosan, or of CHS3, encoding a chitin synthase, or of CSR2, which probably regulates Chs3, all released melanin into the culture supernatant, suggesting a role for chitin or chitosan in retaining the pigment polymer in its normal intracellular location (3, 4). However, vesicles externalized from C. neoformans cells also show laccase activity (21), so the effect of chitin may be on vesicle externalization rather than on melanin itself. Internal structures compatible with mammalian melanosomes have been observed in Cladosporium carrionii (73) and in Fonsecaea pedrosoi (2, 26). Remarkably, F. pedrosoi also secretes melanin and locates the polymer within the cell wall (1, 2, 25, 27, 74).Melanization has been found to play an important role in the virulence of several human fungal pathogens, such as C. neoformans, A. fumigatus, P. brasiliensis, S. schenckii, H. capsulatum, B. dermatitidis, and C. posadasii (among recent reviews are references 29, 42, 62, 74, and 79). From these and earlier reviews of the extensive literature, melanin has been postulated to be involved in a range of virulence-associated properties, including interactions with host cells; protection against oxidative stresses, UV light, and hydrolytic enzymes; resistance to antifungal agents; iron-binding activities; and even the harnessing of ionizing radiation in contaminated soils (15). The most extensively studied fungal pathogen for the role of melanization is C. neoformans, which possesses two genes, LAC1 and LAC2, encoding melanin-synthesizing laccases (52, 69, 90). It has been known since early studies with naturally occurring albino variants of C. neoformans (39) that melanin-deficient strains are attenuated in mouse models of cryptococcosis. Deletion of both the LAC1 and LAC2 genes reduced survival of C. neoformans in macrophages (52), and a study based on otherwise isogenic LAC1⁺ and LAC1⁻ strains confirmed the importance of LAC1 in experimental virulence (66). Other genes in the regulatory pathway for LAC1 are similarly known to be essential to virulence (12, 84).C. albicans has been shown to produce melanin with DOPA as a substrate for production of the polymer (53). The cells could be treated with hot acids to produce typical melanin “ghosts,” and antibodies specific for melanin reacted with the fungal cells by immunohistochemistry with tissues from experimentally infected mice, demonstrating that C. albicans produces melanin in vivo (53). However, no candidate genes encoding laccases have yet been identified in the C. albicans genome (http://www.candidagenome.org/). In this study, we investigated the production of melanin by C. albicans and showed that its normal externalization from wild-type cells, including formation of melanosomes, can be altered to an intracellular and intrawall location by mutation of genes involved in chitin synthesis. C. albicans has four genes encoding chitin synthase enzymes. CHS1 is an essential gene under normal conditions (59), and its product is the main enzyme involved in septum formation (83). Chs3 forms the bulk of the chitin in the cell wall and the chitinous ring at sites of bud emergence (8, 51, 57), while Chs2 contributes to differential chitin levels found between yeast and hyphal forms of the fungus, and Chs8 influences the architecture of chitin microfibrils (43, 51, 55, 57, 58). We found that melanin externalization was unaffected in a chs8Δ mutant but was reduced or abrogated in chs2Δ and chs3Δ mutants. Expression profiles of melanin-producing cells grown in the presence of l-DOPA did not identify any potential laccase-synthesizing genes.