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 chs3Delta and chs2Delta chs3Delta mutants but were fully externalized in chs8Delta and chs2Delta chs8Delta 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.

Fig. 1.

Melanin production by C. albicans SC5314 determined by OD₃₁₀ for whole cultures (a and b) and in resuspended cell pellets (c) in daily samples from cultures with various additions. The points are means of replicate experiments (n = 3 to 6), and the error bars indicate standard errors of the mean. (a) Open squares, cultures with 1 mM DOPA; open triangles, cultures with 1 mM DOPA and 1 mM NAG; open circles, cultures with 1 mM DOPA and 5 mM NAG. (b) Open squares, cultures with 5 mM DOPA; open triangles, cultures with 5 mM DOPA and 1 mM NAG; open circles, cultures with 5 mM DOPA and 5 mM NAG. (c) Mean ± standard deviation (SD) OD₃₁₀ for resuspended pellets of SC5314 cultures sampled at daily intervals. Open squares, cultures with 1 mM DOPA; open triangles, cultures with 1 mM DOPA and 1 mM NAG; open circles, cultures with 1 mM DOPA and 1 mM glucosamine (measured to 4 days only).

Fig. 2.

Transmission electron micrographs of C. albicans SC5314 from cultures after 5 days with no addition of 1 mM DOPA (a) or with addition of 1 mM DOPA sampled after 3 days (b), 4 days (c), and 5 days (d). Bars, 0.5 μm.

Fig. 3.

Scanning electron micrographs of C. albicans SC5314 from cultures after 5 days with no addition of 1 mM DOPA (a) or with addition of 1 mM DOPA (b and c). Bar, 1 μm.

Fig. 4.

Transmission electron micrographs of unstained C. albicans SC5314 cultures after 5 days without DOPA (a) and with addition of 1 mM DOPA (b). The samples were not exposed to heavy metals at any stage of processing of fixation or processing of the sections. (b) The granules assumed to be melanosomes external to the yeast cell surface appear electron dense even without stain. Bars, 0.5 μm.

Fig. 5.

Transmission electron micrographs of C. albicans SC5314 grown for 5 days under control cultures (a) and in cultures containing 1 mM DOPA plus 5 mM NAG (b and c). (b and c) Profuse accumulations of melanosomes are seen surrounding cells grown with DOPA and NAG. Bars, 0.5 μm.

Fig. 6.

High-pressure frozen/freeze-substitution TEM of C. albicans SC5314 grown without DOPA (a and b) and with 1 mM DOPA (c and d). (a and b) In control cells, a fimbriate outer cell wall layer was revealed by this technique. (c and d) In cells with a melanin coat, the melanin particles appeared to adhere to the tips of the hairy strands. Bars, 200 nm (a and c) or 100 nm (b and d).

Fig. 7.

Melanin production by a selection of C. albicans isolates from each of the four major strain clades. The optical densities shown are for supernatants of cultures in DOPA plus NAG after 5 days of incubation. The experiment was performed only once.

Fig. 8.

High-pressure frozen/freeze-substitution TEM of C. albicans chitin synthase mutants grown for 5 days under different melanin-inducing conditions: control, medium containing 1 mM DOPA or medium containing 1 mM DOPA plus 5 mM NAG. (a) chs2Δ chs8Δ control cells. (b) chs2Δ cells grown in DOPA. (c) chs2Δ cells grown in DOPA plus NAG (the arrows indicate a melanin granule embedded within an unidentified intracellular material). (d) chs3Δ cells grown in DOPA. (e and f) chs3Δ cells grown in DOPA plus NAG. (g) chs8Δ cells grown in DOPA. (h and i) chs2Δ chs3Δ cells grown in DOPA. (j) chs2Δ chs3Δ cells grown in DOPA plus NAG. (k) chs2Δ chs8Δ cells grown in DOPA. (l) chs2Δ chs8Δ cells grown in DOPA plus NAG. Bars, 0.5 μm in all micrographs except f and i, where the bars are 200 nm.

Fig. 9.

Transmission electron micrographs with (a) and without (b) heavy metal staining after 5 days of growth in the presence of 1 mM DOPA and 100 μg ml⁻¹ calcofluor white. For comparison, see the control cells in Fig. 2a and 4a. The externalized melanin granules have a different appearance than those in cells grown without calcofluor white (Fig. 2d and 5b). Bars, 0.2 μm.

Fig. 10.

Electron micrographs possibly showing the process of externalization of melanin through the C. albicans SC5314 cell wall. (a and b) Field emission scanning electron micrographs with incompletely externalized melanin granules (bars, 1 μm [a] and 100 nm [b]). (c and d) Conventional TEM (c) and high-pressure frozen/freeze-substituted TEM (d) of cell walls containing probable melanin granules in the process of externalization. Bars, 0.2 μm.