Synthesis of melanin pigment by Candida albicans in vitro and during infection.

Abstract

Melanins are implicated in the pathogenesis of several important human diseases. This study confirmed the presence of melanin particles in Candida albicans in vitro and during infection. Dark particles were isolated from the digestion of C. albicans cultures and from infected tissue, as established by electron microscopy and immunofluorescence techniques.

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INFECTION AND IMMUNITY, Sept. 2005, p. 6147–6150
0019-9567/05/$08.00?0doi:10.1128/IAI.73.9.6147–6150.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 73, No. 9
Synthesis of Melanin Pigment by Candida albicans In Vitro and
during Infection
Rachael Morris-Jones,1* Beatriz L. Gomez,1Soraya Diez,1Martha Uran,2Stephen D. Morris-Jones,3
Arturo Casadevall,4Joshua D. Nosanchuk,5and Andrew J. Hamilton1
Dermatology Department, St. John’s Institute of Dermatology, Guy’s Hospital, Guy’s, Kings, and St. Thomas’ Medical Schools,
London, United Kingdom1; Corporacio ´n para Investigaciones Biolo ´gicas, Medellin, Colombia2; Department of
Microbiology,University College Hospital, London, United Kingdom3; and Department of Microbiology
& Immunology4and Department of Medicine,5Albert Einstein College of Medicine, Bronx, New York
Received 7 February 2005/Returned for modification 15 March 2005/Accepted 4 May 2005
Melanins are implicated in the pathogenesis of several important human diseases. This study confirmed the
presence of melanin particles in Candida albicans in vitro and during infection. Dark particles were isolated
from the digestion of C. albicans cultures and from infected tissue, as established by electron microscopy and
immunofluorescence techniques.
The polymorphic fungus Candida albicans is the causative
agent of candidiasis, which is the most commonly encountered
human fungal disease (3). Despite its global importance, there
is limited information relating to the factors that play a role in
the pathogenesis of candidiasis; however, there is mounting
evidence to suggest that virulence in this organism is multifac-
torial (11). Melanins make up a heterogeneous class of natural
pigments that have a myriad of biological functions (7). Mel-
anization has gained increasing importance as a putative viru-
lence factor in several fungal pathogens and may therefore be
a virulence trait conserved across many fungal species (7). In
this study, we aimed to determine whether C. albicans could
synthesizemelanininyeastcells,utilizingtechniquesdevelopedto
study and isolate melanin from other fungal pathogens. C. albi-
cans strains used were as follows: F28370, F14985, W90572,
W91405, T31119, F10646, and M9958 (University College Hos-
pital, London, United Kingdom); 3179 (National Collection of
Pathogenic Fungi [NCPF], Bristol, United Kingdom); and CIB
1276 and ATCC 24433 (Corporacio ´n para Investigaciones Bio-
lo ´gicas [CIB], Medellin, Colombia). Cryptococcus neoformans
JEC21 (Mel?) and its albino mutant HMC6 (Mel?) were used as
positive and negative controls. Melanin particles were isolated
from yeast cells of all strains of C. albicans and from infected
murine kidney and human skin tissue, as previously described (4),
by sequential digestion steps and boiling in hot concentrated acid.
The resultant particles were a quarter of the size of the yeast cells,
as demonstrated by scanning electron microscopy (Fig. 1A and
B). No particles were isolated from the C. neoformans albino
mutant. Transmission electron microscopy of the melanin parti-
cles from C. albicans revealed small thin layers of electron-dense
material surrounding a void (Fig. 1C and D). Electron spin res-
onance spectroscopy of the dark particles isolated was performed
at Albert Einstein College of Medicine, Bronx, N.Y., by docu-
mented methodology (8, 13). The spectra were identical to the
signals generated from C. neoformans-derived melanin (reference
14 and data not shown).
Enzyme-linked immunosorbent assay plates coated with
melanin (from C. albicans, Sporothrix schenckii, and C. neofor-
mans) were prepared as previously described (6) and incu-
bated with anti-melanin monoclonal antibodies (MAbs) 8B5
generated against S. schenckii yeast melanin (6), 6D2 gener-
ated against melanin from C. neoformans (10), and 8F5 gen-
erated against melanin from Aspergillus fumigatus (15). Nega-
tive controls included wells with no melanin and peroxidase-
conjugated goat anti-mouse (GAM) alone. The anti-melanin
MAbs reacted against the melanin particles derived from C. al-
bicans (Fig. 2), and the reactivities were equal in intensity to those
observed with melanin particles from the positive controls.
C. albicans 3179 yeast cells were embedded in optimal cutting
temperature compound (BDH), and the frozen blocks were sec-
tioned (cryostat Figocut 2700) and stored at ?20°C. Fungal sec-
tions were fixed in cold acetone and air dried. Slides were blocked
with Superblock (Roche, Sussex, United Kingdom) overnight at
4°C and incubated for 2 h at 37°C either with 10 ?g of MAb 8B5,
8F5, or 6D2 with a 1:100 dilution of fluorescein isothyiocyanate
(FITC)GAMimmunoglobulinM(IgM)for2hat37°C.Negative
controls consisted of 5C11 with FITC-labeled antibody or FITC-
labeled antibody alone. Microscopy showed anti-melanin MAb
bound to small structures within the cryosectioned C. albicans
yeast cells (Fig. 3A to C). Uncut yeast did not bind to the anti-
melanin MAbs. Melanin extracted from C. albicans yeast was also
reactive; these particles typically formed aggregates (Fig. 3D and
E). Melanin particles were unreactive with the negative control,
MAb 5C11.
Immunocompetent mice were immunized with C. albicans
F28370. The infection protocol was a modified version of that
previously described (2). At the CIB in Colombia, isogeneic
6-week-old male BALB/c mice were immunized intravenously
via the lateral tail vein with 100 ?l of C. albicans (4 ? 106
CFU/mouse) in sterile phosphate-buffered saline and sacri-
ficed at 21 days. Target organs (heart, lungs, liver, spleen, and
kidneys) were fixed and embedded in paraffin, and sera were
stored at ?70°C. Paraffin-embedded human skin samples from
patients infected with cutaneous C. albicans (a gift from A.
* Corresponding author. Mailing address: St. John’s Institute of
Dermatology, St. Thomas’ Hospital, London SE1 9RT, United King-
dom. Phone: 02079554663. Fax: 011 44 2079552103. E-mail: themojos
@boltblue.com.
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Restrepo, CIB) were also used. Tissue was sectioned and
stained with periodic acid-Schiff stain and methenamine silver
(Grocott modification); blocks positive for fungi were pro-
cessed for immunohistochemical staining with MAbs, as pre-
viously described (6). Briefly, tissue was incubated with mela-
nin-binding MAbs at a dilution of 1:100 at 37°C and then with
1:100 FITC-conjugated GAM IgM. Negative controls were
described above. C. albicans melanin particles were isolated
from the tissues and from cultures and air dried onto 3-amin-
opropyltriethoxysilane slides. Tissues were then probed with
anti-melanin MAbs and FITC GAM IgM, as described above.
C. albicans yeast and hyphae were seen in significant numbers
in the kidneys alone. Reactivity with anti-melanin MAb 8B5
was observed as small fluorescent particles within yeast cells
(Fig. 3F and G) but not in the hyphal forms. Digestion of
infected murine kidneys resulted in isolation of melanin par-
ticles, which reacted with anti-melanin MAb (Fig. 3H and I).
Sera (diluted 1:100) from C. albicans-infected mice showed
positive recognition of the melanin from cultures and tissue
compared to normal mice sera (data not shown). Melanin
FIG. 1. Scanning electron microscopy of Candida yeast cells, before and after treatment with enzyme denaturant and hot acid. C. albicans
F14985 yeast cells (A) and melanin particles (B); bars, 1 ?m. Transmission electron microscopy of C. albicans F14985 melanin particles at low
(C) and high (D) magnification, respectively; bars, 2 ?m.
FIG. 2. Enzyme-linked immunosorbent assay reactivities of anti-
melanin MAbs 7C5 (S. schenckii melanin), 8F5 (A. fumigatus melanin)
and 6D2 (C. neoformans melanin) with melanin particles extracted
from C. albicans isolates (3179 NCPF, F14985, and F28370). Positive
control melanins, S. schenckii (16127) and C. neoformans (JEC21). The
secondary antibody was a 1:1,000 dilution of peroxidase-labeled GAM
(IgM isotype). Negative controls (i.e., wells with no melanin, peroxi-
dase-conjugated GAM alone, and the irrelevant MAb 5C11) are
shown as a combined mean optical density value. Bars are negative
control, 7C5, 8F5, and 6D2, respectively.
FIG. 3. C. albicans (3179 NCPF) yeast cells cryosectioned and stained with anti-melanin MAb 8B5. Bright-field (A) and corresponding
immunofluorescence (B) images of the yeast culture (magnification, ?100) are shown. Bright-field imagery superimposed on immunofluorescence
(C) to show the spatial relationship between the yeast cells and the fluorescent particles (magnification, ?50). Corresponding bright-field (D) and
immunofluorescence (E) microscopy images of C. albicans 3179 NCPF melanin particles isolated from yeast cells reacted with anti-S. schenckii
MAb 8B5 (magnification, ?100) are shown. (F and G) Murine kidney infected with C. albicans F28370, showing fungal hyphae and yeast cells
within the glomerulus. Corresponding bright-field (F) and immunofluorescence (G) images show small fluorescent particles within the yeast cells
(arrows) but not the hyphae when the preparation was stained with anti-S. schenckii melanin MAb 8B5 (magnification, ?40). (H and I)
Corresponding bright-field (H) and immunofluorescence (I) microscopy images showing labeling of C. albicans F28370 aggregated melanin
particles recovered from infected murine kidney with anti-melanin MAb 8B5 (magnification, ?100).
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particles were also isolated from infected human skin tissue
and reacted with the anti-melanin MAbs (data not shown).
Laccase enzymes are utilized in the synthesis of melanin;
hence, their activity was anticipated in C. albicans cytoplasmic
yeast extracts (CYEs). These were harvested as previously
described (13), and protease inhibitor cocktail was added
(Sigma). The suspension was frozen using liquid nitrogen and
smashed until ?90% of the cells were broken. The CYE was
concentrated using Amicon tubes (molecular mass cutoff, 1
kDa), and the protein content was determined by Coomassie
blue methodology. Commercially prepared laccase (Rhus ver-
nificera) (Sigma) and CYEs from C. albicans strains were sep-
arated by 10% polyacrylamide gel electrophoresis (30 mA
overnight) under nondenaturing conditions. Duplicate samples
were loaded onto the gels, one of which had been boiled for 5
min. Gels were incubated with 1 mM L-3,4-dihydroxypheny-
lalanine (L-DOPA) buffer overnight. Positive laccase activity
was revealed by C. albicans CYEs, as shown by dark bands
(Fig. 4), which confirmed L-DOPA had polymerized to form
melanin. Boiling of the samples prior to loading into the gel
eliminated the laccase activity.
This study provides the first definitive evidence that mela-
nization occurs in C. albicans and may represent a new viru-
lence factor. The biosynthesis of melanin in most fungal patho-
gens leads to the accumulation of pigment beneath the cell
wall, resulting in so-called melanin ghosts which retain the
shape and size of the original propagules (9). However, the
small spheres of melanin derived from C. albicans are more
akin to the sclerotic bodies (comparable to melanosomes)
found in Fonsecaea pedrosoi, which causes chromoblastomyco-
sis (1). C. albicans yeast cells secrete complex polymers into
biofilm structures (5) whose composition is not fully under-
stood; one hypothesis therefore might be that melanin parti-
cles are secreted into these resistant extracellular structures.
Melanization may be an attractive target for novel antimicro-
bial drugs; therefore, the investigation of melanin-inhibiting
compounds should be pursued in the future. The genome of C.
albicans has recently been unraveled (12), so evidence from
genetic studies is likely to shed further light on these findings.
We thank the Wellcome Trust (United Kingdom) for supporting
R.M.-J. J.D.N. and A.C. are supported by National Institutes of Health
grant NIH AI52733
We thank A. Restrepo (Corporacio ´n para Investigaciones Biolo ´gi-
cas, Medellin, Colombia) for supplying human skin tissue infected with
candidiasis and Luz Elena Cano for supplying tissue from murine
candidiasis infection models. We thank A. Robson and G. Orchard (St
John’s Institute of Dermatology, London, United Kingdom) for der-
matopathological support, and P. Aisen (Department of Physiology
and Biophysics, Albert Einstein College of Medicine, Bronx, N.Y.) for
help with TEM and SEM studies.
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Editor: T. R. Kozel
FIG. 4. Nonreducing sodium dodecyl sulfate-polyacrylamide gel of
cytoplasmic antigen extract of C. albicans developed with L-DOPA.
Lanes: A, commercial laccase (50 U equivalent); B, 300 ?g of yeast
antigen of C. albicans F28370; C, the same as for lane B but boiled for
5 min; D, 300 ?g of yeast antigen of C. albicans F14985; E, the same
as for lane D but boiled for 5 min; F, 300 ?g of yeast antigen C.
albicans NCPF 3179; G, the same as for lane F but boiled for 5 min.
6150NOTESINFECT. IMMUN.