Melanin Externalization in Candida albicans Depends on Cell Wall Chitin Structures

Abstract

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 chs3⌬ and chs2⌬ chs3⌬ mutants but were fully externalized in chs8⌬ and chs2⌬ chs8⌬ 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 foun...

Activation of Melanin Synthesis in Alternaria infectoria by Antifungal Drugs

“…This is in accordance with our previous observations that, at the nikkomycin Z concentration used in the present study, the cell wall chitin levels increased in relation to those for control cells in this strain of A. infectoria (40). The same was observed in Wangiella dermatitidis, C. albicans, and C. neoformans, indicating that cell wall chitin is a scaffold required to cross-link melanin to the cell wall components (36,63,64). On the other hand, TEM revealed that the electron-dense well-defined outer layer of the cell wall is disturbed when the fungus was exposed to nikkomycin Z, an inhibitor of chitin synthases.…”

“…On the other hand, TEM revealed that the electron-dense well-defined outer layer of the cell wall is disturbed when the fungus was exposed to nikkomycin Z, an inhibitor of chitin synthases. We believe that this change in the organization of the melanin is related to the anchoring of melanin to chitin microfibrils, as described previously (36). In fact, although nikkomycin Z does not decrease the total amount of chitin (40), it is possible that it contributes to a different organization of the chitin microfibrils, thus leading to changes in the pattern of melanin deposition, as described in C. albicans (36).…”

“…Accordingly, it was previously suggested that melanization may be a mechanism for the acquisition of resistance to polyenes and echinocandins in Cryptococcus neoformans (50). In C. albicans it was described that deposition of the cell wall melanin is dependent on chitin synthesis (36). As such, here we describe that nikkomycin Z leads to increased melanization.…”

“…We believe that this change in the organization of the melanin is related to the anchoring of melanin to chitin microfibrils, as described previously (36). In fact, although nikkomycin Z does not decrease the total amount of chitin (40), it is possible that it contributes to a different organization of the chitin microfibrils, thus leading to changes in the pattern of melanin deposition, as described in C. albicans (36). Moreover, on the basis of what is known for other fungi, melanin is a powerful immunomodulatory factor contributing to protecting fungal cells from oxidative burst and other host immune mechanisms.…”

“…After centrifugation, the pigment was recovered from the supernatant and the pellet was extracted again until no more extractable pigment was obtained (32,34). For quantification of pigment from A. infectoria, no more steps of purification were undertaken to avoid pigment loss, and the pigment concentration was measured photometrically in a Spectra Max Plus384 spectrophotometer (Molecular Devices, LLC) at a wavelength of 340 nm (35,36).…”