Ultrastructural characterization of melanosomes of the human

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Journal of Structural Biology 162 (2008) 75–84

StructuralBiology

Ultrastructural characterization of melanosomesof the human pathogenic fungus Fonsecaea pedrosoi

Anderson J. Franzen a,b, Marcel M.L. Cunha a, Kildare Miranda c, Joachim Hentschel d,Helmut Plattner d, Moises B. da Silva e, Claudio G. Salgado e, Wanderley de Souza c,

Sonia Rozental a,*

a Laboratorio de Biologia Celular de Fungos, Instituto de Biofısica Carlos Chagas Filho (IBCCF), Universidade Federal do Rio de Janeiro (UFRJ),

Rio de Janeiro, Brazilb Laboratorio de Tecnologia em Bioquımica e Microscopia, Centro Universitario da Zona Oeste, Rio de Janeiro, Brazil

c Laboratorio de Ultraestrutura Celular Hertha Meyer, IBCCF, UFRJ, Rio de Janeiro, RJ, Brazild Fachbereich Biologie, Universitat Konstanz, Konstanz, Germany

e Laboratorio de Dermato-Imunologia, Universidade Estadual do Para, Universidade Federal do Para, Marcello Candia, Belem, Brazil

Received 14 July 2007; received in revised form 26 October 2007; accepted 9 November 2007Available online 22 November 2007

Abstract

Melanin is a complex polymer widely distributed in nature and has been described as an important virulence factor in pathogenicfungi. In the majority of fungi, the mechanism of melanin formation remains unclear. In Fonsecaea pedrosoi, the major etiologic agentof chromoblastomycosis, melanin is stored in intracellular vesicles, named melanosomes. This paper details the ultrastructural aspects ofmelanin formation, its storage and transportation to the cell wall in the human pathogenic fungus F. pedrosoi. In this fungus, melaninsynthesis within melanosomes also begins with a fibrillar matrix formation, displaying morphological and structural features similar tomelanosomes from amphibian and mammalian cells. Silver precipitation based on Fontana-Masson technique for melanin detection andimmunocytochemistry showed that melanosome fuses with fungal cell membrane where the melanin is released and reaches the cell wall.Melanin deposition in the fungal cell wall occurs in concentric layers. Antibodies raised against F. pedrosoi melanin revealed the sites ofmelanin production and storage in the melanosomes. In addition, a preliminary description of the elemental composition of this orga-nelle by X-ray microanalysis and elemental mapping revealed the presence of calcium, phosphorus and iron concentrated in its matrix,suggesting a new functional role for these organelles as iron storage compartments.� 2007 Elsevier Inc. All rights reserved.

Keywords: Fonsecaea pedrosoi; Melanin; Cell wall; Melanosome; X-ray microanalysis

1. Introduction

Melanins are phenolic and heterogeneous biopolymers,produced and distributed among a wide range of organ-isms, which plays a role between protection and virulence,and possibly contributes to the maintenance of several spe-cies along evolution (Plonka and Grabacka, 2006). In path-ogenic fungi, the synthesis and expression of these

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doi:10.1016/j.jsb.2007.11.004

* Corresponding author. Fax: +55 21 22808193.E-mail address: [email protected] (S. Rozental).

pigments have been associated with microbial virulence(Gomez et al., 2001). Melanized fungi have been shownto be more resistant to host defenses. Several features ofmelanin appear to be involved in fungal resistance, suchas scavenging of free radicals, capacity to interact withantifungal drugs preventing them from reaching their tar-get sites, absorption of UV light, protection against enzy-matic lysis, desiccation and extreme variations oftemperature (Butler and Day, 1998). Due to its complexity,difficulty extraction/isolation, and diversity, the exactstructure and composition of any melanin is still unknown.

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76 A.J. Franzen et al. / Journal of Structural Biology 162 (2008) 75–84

In fungi, the study of melanin synthesis, storage and trans-port could potentially provide important insights for therational development of active drugs that could positivelyinterfere with this strong microbial virulence factor(Nosanchuk and Casadevall, 2003).

In mammalian cells, the biosynthesis of melanin occursthrough a cation-dependent mechanism in unique mem-brane-enclosed structures of melanocytes and retinal pig-mented epithelial cells named melanosomes (Salceda andRiesgo-Escovar, 1990). The mammalian melanogenesisprocess begins with the formation of premelanosomes,vesicular structures with internal membranes without mel-anin (stage I). This organelle differentiates into elongatedstructures with internal striations (stage II). Melanin isthen accumulated within the striations, resulting in theirthickening and blackening (stage III) until this pigment fillsthe entire melanosome (stage IV). The melanosomes arethen either stored or secreted and incorporated by othercell types, such as the keratinocytes (Seiji et al., 1963a,b;Marks and Seabra, 2001).

The in vitro and in vivo production of melanin wasrecently demonstrated in important human pathogenicdimorphic fungi such as Blastomyces dermatitidis (Nosan-chuk et al., 2004), Paracoccidioides brasiliensis (Gomezet al., 2001), Sporothrix schenckii (Morris-Jones et al.,2003), Penicillium marneffei (Youngchim et al., 2005), His-

toplasma capsulatum (Nosanchuk et al., 2002) and Coccid-

ioides posadasii (Nosanchuk and Casadevall, 2006).In fungal pathogens, melanin biosynthesis is usually

described as occurring at the cell wall and being derivedfrom phenolic compounds or intermediates. However,Alviano et al. (1991) showed that in the black fungi Fonse-

caea pedrosoi, melanin is synthesized within membranebounded structures referred to as melanosomes. Similarorganelles were described in other black fungi, such asCladosporium carrioni and Hormoconis resinae (San-Blaset al., 1996).

Fonsecaea pedrosoi is a dematiaceous and medicallyimportant pathogenic fungus in tropical countries and isthe most frequent etiologic agent of chromoblastomycosis,an endemic subcutaneous mycosis commonly observed inthe Amazon region (Bonifaz et al., 2001; Silva et al.,1999). It produces melanin constitutively and has been con-sidered as a model of melanin-producing fungi (Alvianoet al., 2004; Cunha et al., 2005; Franzen et al., 1999,2006). In contrast to other fungal species that need externalprecursors (L-Dopa, e.g.) to synthesize this pigment (Chas-kes and Tyndall, 1975; Kojeg et al., 2004; Williamson,1994), F. pedrosoi synthesizes melanin constitutivelythrough the polyketide pathway, a process that starts withacetyl-CoA (Jacobson, 2000).

The ability of melanin to bind transition metals and toquench free radicals is thought to be an important factorof virulence (Jacobson, 2000). Iron is essential for oxida-tion-reduction reactions (Byers and Arceneaux, 1998) andhas been previously described as an important constituentof fungal melanin and responsible for their protection

against oxidative mechanisms (Henson et al., 1999). Inmicroorganisms, acidic conditions are necessary for suchmetals to bind to polyphosphate (Docampo et al., 2005).The production of melanin inside melanosomes of F.

pedrosoi occurs in an acidic environment, as previouslyrevealed by the accumulation of acridine orange insidethese organelles (Franzen et al., 1999). The estimatedintra-melanosomal pH in mammalian cells is around 3–5,melanin biosynthesis is reduced at neutral pH as majorenzymes involved in melanogenesis have no activity at thispH (Bathnagar et al., 1993).

Calcium is a ubiquitous intracellular second messengerthat regulates a wide range of cellular activities (Berridgeet al., 2000). The accumulation of calcium and melanin inanimal melanosomes suggests that melanin granules couldperhaps serve as a calcium reservoir (Salceda and Riesgo-Escovar, 1990; Salceda and Sanchez-Chavez, 2000).

In this report, the ultrastructure of melanosomes of F.

pedrosoi was further analyzed by transmission electronmicroscopy. Analysis of thin sections showed the fourstages of melanogenesis in the melanosomes of F. pedrosoi,sharing similar features of amphibian and mammalian mel-anosomes described by Prelovsek and Bulog (2003) andRaposo and Marks (2002). Preliminary analysis of the ele-mental content of the melanosomes by electron probe X-ray microanalysis and elemental mapping showed the pres-ence of phosphorus, iron and calcium concentrated in themelanosomal matrix. Interestingly, the same elements havebeen found in the cell wall. Cytochemical and immunocyto-chemical detection of melanin showed the concentration ofthis polymer in the melanosomes and in the cell wall, whereit was organized in concentric layers. In addition, transportand release of melanosomal content to the cell wall,through a fusion mechanism of the melanosomes with theplasma membrane, was observed. Altogether, the resultscontribute for the further understanding of the functionalrole of this organelle in pathogenic fungi.

2. Materials and methods

2.1. Chemicals

The majority of the reagents and organic solvents werepurchased from Merck (Rio de Janeiro, RJ, Brazil).Gold-labeled goat anti-human IgG antibody was pur-chased from Pelco (Redding, CA, USA).

2.2. Microorganism and growth conditions

A human isolate of F. pedrosoi (strain 5VLP, Oliveiraet al., 1973) was used in the present work. Stock cultureswere maintained on Saubouraud’s glucose agar mediumunder mineral oil and kept at 4 �C. Conidia were obtainedfrom the stock culture and incubated at 28 �C for 5 days inCzapeck-Dox modified medium (CDM, each indicated asg · L�1: sucrose 30, NaNO3 2, KH2PO4 1, MgSO4Æ7H2O0.5, KCl 0.5, ammoniacal iron citrate 0.01), under shaking

A.J. Franzen et al. / Journal of Structural Biology 162 (2008) 75–84 77

conditions. The culture was filtered in a 40–60 G porousplate filter to retain mycelia whereas conidia passedthrough the filter. Cells were then recovered by centrifuga-tion (13.600g, 30 min, 4 �C).

2.3. Transmission electron microscopy (TEM)

Cells were fixed with 2.5% glutaraldehyde and 4% para-formaldehyde with 10 mM calcium chloride added in100 mM cacodylate buffer, pH 7.4, for 1 h at 25 �C, washedin cacodylate buffer and then post-fixed with 1% OsO4 and0.8% K4Fe(CN)6Æ3H2O in 100 mM cacodylate buffer for2 h. Samples were then washed in cacodylate buffer, dehy-drated in graded series of acetone and embedded in Spurrresin. Ultrathin sections were routinely stained with aque-ous uranyl acetate and alkaline lead citrate and examinedin a TEM type JEOL 1200 EX, operating at 80 kV.

2.4. Electron probe X-ray microanalysis and elemental

mapping

To investigate the elemental composition of F. pedrosoi

melanosomes, cells were grown in presence or in absence of0.01 g · L�1 ammoniacal iron citrate and processed forTEM analysis by fixation with 2.5% glutaraldehyde and4% paraformaldehyde with 10 mM calcium chloride addedin 100 mM cacodylate buffer, pH 7.4, for 1 h at 25 �C,washed in cacodylate buffer and then dehydration ingraded series of acetone and embedding in Spurr resin.Ultrathin sections of 70 nm were examined in a TEM typeJEOL 1200 EX at 80 kV using a low background carbonholder. X-rays were collected for 300 s using a Li-driftedSi detector with Norvar window in a 0 to 10 KeV energyrange with a resolution of 10 eV/channel. Samples weretilted 30� before the analysis. Analyses were performedusing a Noran/Voyager III analyzer. To prevent and/orminimize ion extraction from the melanosome during rou-tine preparation, unfixed whole conidia cells (no fixativesand staining agents added) were air-dried onto formvarcoated grids and observed in an energy filtering TEM asdescribed elsewhere (Miranda et al., 2004a,b; Medeiroset al., 2005). Cells cultivated in the presence of Fe3+ addedas chloride, pH 7.2, were resuspended in phosphate buffersaline (PBS), droplets were applied to 100 mesh formvar-coated copper grids, allowed to adhere for 10 min, carefullyblotted dry and observed in a LEO EM 912 electron micro-scope. Electron spectroscopic images were recorded fromwhole cells at an energy loss of �60 eV with a spectrometerslit width of 20 eV. The energy filter was used only with thepurpose of filtering out the chromatic aberration caused bythe interaction of the electron beam with thick specimen(whole cells) and to improve the contrast between the cyto-plasm and intracellular structures (contrast tuning). Ele-ment-specific images were not obtained with the energyfilter. Energy dispersive X-ray spectra were recorded fromthe melanosomes of whole cells dried onto formvar coatedgrids. Control spectra were collected from cell cytoplasm

and from the formvar film. X-rays were collected for200 s using a Li-drifted Si-detector (front area 30 mm2)equipped with an atmospheric thin window (ATW). Sam-ples were not tilted and a low background titanium gridholder was used. The microscope was operated at 80 kVusing a tungsten filament, in the scanning transmission(STEM) imaging mode, spot size was 40 nm. Analyses wereperformed using a Link multichannel energy analyzer andLink ISIS 3.00 software (Oxford Instruments Wiesbaden,Germany). For elemental mapping, 400 frames wereacquired with a dwell time of 100 ls per pixel with a reso-lution of 512 · 512 pixels (total of 2.9 h). All element-spe-cific maps were drift corrected and backgroundsubtracted using specific tools within Inca program.

2.5. Cytochemistry for melanin detection

Cells were fixed with 1% glutaraldehyde (EM grade) indistilled water and then processed for Fontana-Masson sil-ver staining used for histochemical studies in light micros-copy, but adapted to TEM (Kimura and McGinnis, 1998;Taborda et al., 1999). The technique is based on a silversolution made of 10% silver nitrate in distilled water fol-lowed by the addition of ammonium hydroxide until a faintopalescence appears. The ammoniated silver nitrate solu-tion is mixed with distilled water, filtered and stored over-night at 25 �C in the dark. Conidia were incubated for30 min in this solution and then washed three times inwater and fixed again with 1% glutaraldehyde and 4%paraformaldehyde in water. Cells were then washed inwater, dehydrated in graded series of acetone, embeddedin Spurr resin. Ultrathin sections of 70 nm were examinedin a TEM, type JEOL 1200 EX, operating at 80 kV.

2.6. Immunocytochemistry for melanin detection

Cells were fixed with 0.2% glutaraldehyde (grade I), 1%picric acid and 4% paraformaldehyde with 10 mM calciumchloride added in 100 mM cacodylate buffer, pH 7.4, for1 h at 25 �C washed in PBS with 3% of bovine serum albu-min, dehydrated in graded series of ethanol at 4 �C andembedded in Unicryl resin at �20 �C. Polymerization wascarried out at �20 �C under UV radiation for 96 h. Ultra-thin sections were collected on nickel grids, incubated in80 mM ammonium chloride in PBS for 30 min to quenchfree aldehyde groups and transferred to PBS supplementedwith 0.01% Tween 20, 1.5% bovine serum albumin, 0.5%fish gelatine, pH 7.4 (blocking buffer) for 1 h at room tem-perature. Grids were subsequently incubated for 1 h in thepresence of anti-melanin (F. pedrosoi) polyclonal antibody(1:20 dilution), purified from the sera of human patients aspreviously described (Alviano et al., 2004). The sampleswere then washed twice with PBS-BSA and incubated for1 h in the presence of gold-labeled (10 nm particle size)goat anti-human IgG (1:100 dilution) secondary antibody.As a control, incubation with the primary antibody againstmelanin was omitted. The sections were then successively


washed in PBS and water, stained in aqueous uranyl ace-tate and alkaline lead citrate and observed in a TEM typeJEOL 1200 EX.

3. Results

3.1. Melanogenesis in F. pedrosoi

Thin sections of F. pedrosoi (Fig. 1A–D) showed thepresence of a fibrillar matrix and the gradual depositionof melanin inside the melanosomes. Their appearances var-

Fig. 1. Ultrathin sections of Spurr embedded F. pedrosoi conidia revealing nummitochondria (m) and fungal cell wall (cw). Structures corresponding to fMelanosomal fibrillar matrix can be clearly observed (G–J) due to its increasingis followed by melanin deposition in the periplasmic space of the cell wall (ar

ies, but generally were observed as electron lucent mem-brane-enclosed organelles with electron dense content ofdifferent profiles of melanin deposition. The first step isthe formation of a fibrillar matrix (arrow in Fig. 1A; highermagnification in Fig. 1G) with a subsequent stage of fibril-lar matrix thickening (Fig. 1B and H). The darkening ofthe fibril and the partial filling of the granule were revealedby the local increase in electron density in the middle of thegranule, probably due to melanin deposition (Fig. 1C, highmagnification in Fig. 1I). The later stage could be observedwhen the granule became completely electron dense

erous melanosomes (M) at different stages of maturation (arrows in A–D),our different stages of melanin granules deposits are indicated (G–J).electron density. Melanosomes fusion with fungal membrane (arrow in E)

row in F). Bars A–F = 0.2 lm; G–J = 0.1 lm.


(Fig. 1D and J). The mature melanosome were directed tothe cell membrane for fusion (arrow in Fig. 1E) and therelease of their content towards the cell wall (arrow inFig. 1F).

3.2. X-ray microanalysis of the melanosomes of F. pedrosoi

For a preliminary characterization of the elementalcomposition of the melanosomes by X-ray microanalysis,thin sections (where the melanosomes are easily recog-nized) of cells cultivated in the presence and absence of ironwere used. From the structural point of view, cells culti-vated in the presence of iron apparently had melanosomeswith higher electron density (Fig. 2A) than cells grown inthe absence of iron (Fig. 2D). Preliminary examinationby X-ray microanalysis showed considerable amounts ofphosphorus and calcium within the melanosome (Fig. 2Band E). Small iron peaks were detected in cells grown inthe presence of iron salt whereas no iron signal wasdetected in melanosome of cells cultivated in absence ofthis cation (Fig. 2E). Using this method, except for residualphosphorus from the buffer, no characteristic peaks foundin the melanosomes were detected in cytoplasmic regions(Fig. 2C and F). Copper peaks came from grids used forTEM. In order to minimize the mobilization of unboundand diffusible ions possibly extracted by glutaraldehyde/paraformaldehyde fixation and the subsequent steps forroutine preparation of thin sections for TEM (dehydration,

Fig. 2. Energy dispersive X-ray spectrum of thin section of conidia cells from aD) The TEM images of the conidia. (B and E) The elemental profile of the m(arrow in B). The elemental profile of the cytoplasm is presented in (C) and (

embedding and sectioning, e.g.), air-dried unfixed wholecells were used and imaged with an energy-filtered TEM(Fig. 3). In this preparation, phosphorous, calcium andiron (Fig. 3B–D) were found in electron dense organelles,which we presume to correspond to melanosomes, and inthe cell wall. As no significant amounts of these ions couldbe detected in the cytoplasm, it is possible that the elementswere, at least, partially retained in the melanosomes andthe cell wall. Higher magnification of the electron denseorganelles (Fig. 3I) revealed co-localization of phospho-rous, iron and calcium (Fig. 3J–L). Spectra from melano-somes obtained from air-dried whole cells showed largeriron peaks (white arrow in Fig. 3G) then those obtainedfrom thin sections (Fig. 2B). Peaks of sodium and chloridemight correspond to residues from the buffer (Fig. 3G). Nosignificant amounts of the elements found in the melano-some could be detected in the cytoplasmic regions(Fig. 3H) and neither in the formvar film (not shown).

3.3. Cytochemistry for melanin detection

Observation of silver stained sections of F. pedrosoi

revealed the presence of circular and electron dense depositson the cell wall (arrow in Fig. 4A) and in melanosomes(arrowheads in Fig. 4A). The white arrowhead in Fig. 4Ashows the melanin deposition in the periplasmic space ofthe fungal cell, between the cellular membrane and the cellwall. The deposits of silver along the cell wall were orga-

culture supplemented with iron (A–C) or one without iron (D–F). (A andelanosomes. Iron supplemented cells contain iron in their melanosomes

F), neither one shows any iron or calcium signals. Bars = 2 lm.

Fig. 3. Contrast tuned images of F. pedrosoi whole conidia (A) and its corresponding elemental images (B–F) by X-ray microanalysis showing thedistribution of phosphorous (B), calcium (C), iron (D), and oxygen (E). To rule out the potential incorrect detection of signals due to mass thicknesseffects, a continuum map from a peak free region was displayed in (F). Note that the melanosome and cell wall regions did not display significantly highersignals when compared to the other regions of the cell. (G and H) Correspond to X-ray spectra obtained from a melanosome and a cytoplasmic portion,respectively. Note the presence of well defined phosphorus, calcium and iron peaks in the melanosome. (I) Contrast tuned image of an intracellularmelanosome and its corresponding phosphorous (J), calcium (K), iron (L) signals. All element-specific maps were background subtracted. Bars A = 1 lm;I = 0.2 lm.


Fig. 4. Ultrathin sections of F. pedrosoi incubated with AgNO3 revealedsilver deposition on the cell wall (arrow in A) and in intracellularorganelles (arrowheads in A) resembling melanosomes. A deposit ofmelanin could be observed in the periplasmic space of the fungal cell wall(white arrowhead in A). Mature conidia present four distinctive cell walllayers (arrows in B). X-ray spectrum (C) of F. pedrosoi melanosome andcell wall confirm the presence of the element silver. Control cells withoutsilver staining are shown in (D). Bars = 0.5 lm.


nized into 2–4 layers and arranged in a concentric pattern(Fig. 4B). The presence of silver in the electron dense depos-its was confirmed by X-ray microanalysis (Fig. 4C). Copperpeaks are due to the specimen grid used for TEM analysis.Phosphorus, calcium and iron, previously detected in mela-nosomes and cell wall, were not detected probably becausethey were washed out during the cytochemical procedure.Control cells were not labeled (Fig. 4D).

3.4. Immunolocalization of melanin

Gold-labeled (10 nm particle size) goat anti-human IgGwas found in the melanosomes as well as along the wholeextension of the cell wall (Fig. 5). Small peripheral vesicles,gold-labeled, were found in the cell periphery, probablyrepresenting secretion vesicles for exocytosis of the melan-osomal content (Fig. 5B). The electron dense portion of themelanosome was particularly intensely labeled (Fig. 5C).Control cells that were only incubated with gold-labeledgoat anti-human IgG but not with antibodies against mel-anin showed no gold particles.

4. Discussion

A significant advance in the knowledge of fungal mela-nins has been achieved in the last decade, especially regard-ing the molecular aspects of their biosynthesis andimportance for fungal pathogenicity. However, there arefew reports on the production site of melanin in humanpathogenic fungi. Fungal melanins have been generallyfound as part of the cell wall, as an enmeshed polymerwithin the cell wall or incorporated on its outermost layer,often recognizable as a distinct and fairly sharply definedoutside layer (Jacobson, 2000; Langfelder et al., 2003;Nosanchuk and Casadevall, 2003). Our previous studiesshowed that the use of tricyclazole, a specific inhibitor oftwo reductase steps of the polyketide pathway in F. pedro-

soi, induced morphological alterations in the cell wall thatconsequently increase the susceptibility of the fungus tomechanical lyses and destruction by macrophages (Cunhaet al., 2005; Franzen et al., 2006).

Many fungi have been described as possessing melaninexclusively in the cell wall, such as Colletotrichum lagenarium

and Cryptococcus neoformans (Eisenman et al., 2005; Kuboand Furusawa, 1986; Takano et al., 1997), where melaninwas found in layers within the cell wall. In S. schenckii andVerticillium dahliae melanin was seen as granular depositsat the surface of the cell wall (Butler and Day, 1998; Romer-o-Martinez et al., 2000). Previous studies on F. pedrosoi sug-gested that melanin is partly deposited on the cell wall andmostly in cytoplasmic organelles resembling mammalianmelanosomes (Alviano et al., 1991; Franzen et al., 1999).Similar membrane-enclosed structures have also beenobserved in other dematiaceous fungi such as C. carrioni

and H. resinae (San-Blas et al., 1996). In the present work,the use of low viscosity Spurr embedding resin improvedthe visualization of the ultrastructure of the fungal melano-some by TEM. The melanin granules were shown to beformed over a fibrillar matrix—a process not described sofar in fungal cells. In mammalian cells, melanosomes andtheir precursors can be classified in four stages of develop-ment based on morphology (Seiji et al., 1963a,b). Premela-nosomes lack melanin and consist of vesicular structureswith internal membranes (stage I) or characteristic, elon-gated structures with internal striations of unknown compo-sition (stage II). As polymerized melanins are synthesized

Fig. 5. Immunolocalization of melanin in Unicryl thin sections that were gold-labeled with anti-melanin antibodies. M1 and M2 represent two distinctlylabeled melanosomes (A and C). All fungal cell wall layers were labeled with anti-melanin antibodies (A). Labeled vesicles could be seen (arrow in B)fusing with the plasma membrane and depositing melanin on the periplasmic space of the cell wall. Bars A = 0.2 lm; B and C = 0.1 lm.


they accumulate on the striations, resulting in their thicken-ing and blackening (stage III) until melanin fills the entiremelanosome (stage IV) (Raposo et al., 2001). In amphibian,the liver melanosomes also contain a fibrillar structure that isorganized in small circular structures and electron denseclusters dispersed inside the melanosome (Prelovsek andBulog, 2003). The same pattern of clusters was observed inthe present study for F. pedrosoi, but the orientation of thefibrillar matrix follows the parallel array found in mamma-lian melanosomes. The maturation of the clusters continueswith the increase of the deposition of melanin over the fibril-lar matrix until completion of organelle biogenesis.

Melanin binds metals (Fogarty and Tobin, 1996) andfungal melanin and polyphenolic compounds are knownto combine with silver salts which are then reduced to anelectron dense, metallic, state (Kwon-Chung et al., 1981).In black fungi, even though some melanin can be detectedin hematoxylin–eosin stained tissue sections, the Fontana-Masson stain enhances the visualization of this heteroge-neous compound (Wood and Russel-Bel, 1993) and a cop-per sulfide-silver reaction was used for melanin detection infungi and melanocytes (Butler et al., 2005). Silver stainingalso demonstrated the presence of up to four concentriclayers of melanin in the cell wall. This layered formationwas recently described in melanin ghosts of C. neoformans

where the same concentric labeling pattern was visualizedby TEM and described as being composed of packed gran-ules of melanin (Eisenman et al., 2005). In our samples, anincreasing size of silver granules toward the outside of themulticoncentric layers was also observed, suggesting theaccumulation of melanin in the cell wall.

Several authors recently showed that anti-melanin anti-bodies are able to recognize melanin on fungal cell walls(Carzaniga et al., 2002; Rosas et al., 2000; Gomez et al.,2001; Morris-Jones et al., 2003; Nosanchuk et al., 2004;Youngchim et al., 2004, 2005). The immunocytochemistryresults, shown in Fig. 5, further support the cytochemicalresult presented in Fig. 4. Again immunocytochemistrylocalizes the melanin polymer in different melanosomes,and all over the fungal cell wall, confirming that whathas been detected cytochemically is melanin. In addition,the application of anti-melanin antibodies to thin sectionsof F. pedrosoi revealed the fusion of vesicles, containingmelanin, with the fungal plasma membrane and the depo-sition of their content into the cell wall.

According to the exocytosis theory, mammalian melanintransfer is accomplished by fusion of the melanosomalmembrane with the melanocyte plasma membrane, result-ing in the secretion of melanin in a process considered tobe exocytosis (Van den Bossche et al., 2006).

Preliminary examination of the elemental composition ofthe melanosomes by energy dispersive X-ray microanalysisshowed the possible presence of phosphorous, calcium andiron within this organelle, detected both in thin sections andin whole cell mounts. Iron has been previously described asan important constituent of fungal melanin, and responsiblefor the protection against oxidative mechanisms (Hensonet al., 1999). It is possible that the melanosome of F. pedro-

soi participate actively in the cellular physiology of iron, notonly as co-factor of melanin reductase but also as a constit-uent of the melanin molecule. Most of the elements detectedin the melanosomes were also found in the cell wall, what is


in agreement with the hypothesis that melanosomes fusewith the plasma membrane, releasing their content (melaninand cations) in the cell wall and corroborated by the factthat melanin has been previously shown to be an importantcomponent of this structure in some pathogenic fungi(Franzen et al., 2006).

Calcium is an important second messenger that may reg-ulate a wide range of cellular activities (Berridge et al., 2000).Saccharomyces cerevisiae has a simple Ca2+-signaling sys-tem regulating the cell cycle, mating, sensing of glucoseand glucose starvation, resistance to salt stress and cell sur-vival (Zelter et al., 2004). In filamentous fungi, where growthand development are more complex, there is evidence of theinvolvement of Ca2+ in additional physiological processes,including cell cycle regulation, sporulation, spore germina-tion, hyphal tip growth, hyphal orientation, hyphal branch-ing, and circadian rhythms (Shaw and Hoch, 2001). In F.

pedrosoi, calcium is relevant for signals driving the differen-tiation of conidia, and of hyphae to sclerotic cells, the para-sitic form of the fungus (Franzen et al., 2006; Mendoza et al.,1993; Silva et al., 2002). Our results suggest that F. pedrosoi

melanosomes could serve as a calcium reservoir, just likemammalian melanosomes (Salceda and Riesgo-Escovar,1990; Salceda and Sanchez-Chavez, 2000). Calcium storagepossibly occurs as phosphates, as described for the acidocal-cisomes found in several eukaryotic microorganisms (Doc-ampo et al., 2005; Miranda et al., 2004a).

In conclusion, the melanosomes of F. pedrosoi possess afibrillar matrix, similar to that found in the melanosomesof amphibians, functioning as a support site for melanindeposition. Melanin is exported inside this organelle tothe cell wall and is deposited in concentric layers, asdetected by silver staining. F. pedrosoi melanin demon-strates high affinity for iron. In addition, calcium and phos-phorous appear associated with iron and melanin withinthe melanosomes. These results represent the first descrip-tion of such high amounts of those elements in melano-somes what opens new perspectives for the understandingof the functional role of melanin in pathogenic fungi.

Acknowledgments

The authors thank Dr. Marcio Lourenco Rodrigues forhelping in the isolation of human antibodies. The presentwork was supported by grants from Conselho Nacionalde Desenvolvimento Cientıfico e Tecnologico (CNPq) (toS.R. and W.S.), Fundacao Universitaria Jose Bonifacio(FUJB) (to S.R.), Programa de Nanociencia e Nanotecno-logia MCT-CNPq (to K.M.), SCTIE-Ministerio da Saudedo Brasil (to C.G.S.) and the Deutsche Forschungsgeme-inschaft (to H.P.).

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at doi:10.1016/j.jsb.2007.11.004.

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Ultrastructural characterization of melanosomes of the human