Melanin is an essential component for the integrity of the cell wall of Aspergillus fumigatus conidia
© Pihet et al; licensee BioMed Central Ltd. 2009
Received: 23 December 2008
Accepted: 24 August 2009
Published: 24 August 2009
Aspergillus fumigatus is the most common agent of invasive aspergillosis, a feared complication in severely immunocompromised patients. Despite the recent commercialisation of new antifungal drugs, the prognosis for this infection remains uncertain. Thus, there is a real need to discover new targets for therapy. Particular attention has been paid to the biochemical composition and organisation of the fungal cell wall, because it mediates the host-fungus interplay. Conidia, which are responsible for infections, have melanin as one of the cell wall components. Melanin has been established as an important virulence factor, protecting the fungus against the host's immune defences. We suggested that it might also have an indirect role in virulence, because it is required for correct assembly of the cell wall layers of the conidia.
We used three A. fumigatus isolates which grew as white or brown powdery colonies, to demonstrate the role of melanin. Firstly, sequencing the genes responsible for biosynthesis of melanin (ALB1, AYG1, ARP1, ARP2, ABR1 and ABR2) showed point mutations (missense mutation, deletion or insertion) in the ALB1 gene for pigmentless isolates or in ARP2 for the brownish isolate. The isolates were then shown by scanning electron microscopy to produce numerous, typical conidial heads, except that the conidia were smooth-walled, as previously observed for laboratory mutants with mutations in the PKSP/ALB1 gene. Flow cytometry showed an increase in the fibronectin binding capacity of conidia from mutant isolates, together with a marked decrease in the binding of laminin to the conidial surface. A marked decrease in the electronegative charge of the conidia and cell surface hydrophobicity was also seen by microelectrophoresis and two-phase partitioning, respectively. Ultrastructural studies of mutant isolates detected considerable changes in the organisation of the conidial wall, with the loss of the outermost electron dense layer responsible for the ornamentations seen on the conidial surface in wild-type strains. Finally, analysis of the conidial surface of mutant isolates by atomic force microscopy demonstrated the absence of the outer cell wall rodlet layer which is composed of hydrophobins.
These results suggest that, in addition to a protective role against the host's immune defences, melanin is also a structural component of the conidial wall that is required for correct assembly of the cell wall layers and the expression at the conidial surface of adhesins and other virulence factors.
Aspergillus fumigatus, the most common agent of human and animal aspergillosis, is an opportunistic mould responsible for various infections in receptive hosts, ranging from colonisation of the airways in patients with cystic fibrosis to severe and often fatal disseminated infections in immunocompromised patients .
Origin of the mutant isolates studied
(patient with cystic fibrosis)
Susceptibility to dihydroxy-naphtalene (DHN)-melanin inhibitors and characterisation of the genetic defect
Growth on Czapek medium supplemented with inhibitors of melanin biosynthesis
Strain or isolate number
31.7 ± 1.52
30 ± 4.36
29.3 ± 2.08
32.3 ± 0.58
32 ± 2
31.7 ± 1.15
28 ± 1*
31.2 ± 0.28
33.7 ± 0.58
32 ± 2
31 ± 1*
33.3 ± 1.15
31.7 ± 1.15
30.7 ± 1.53
34 ± 1.73
25.3 ± 1.53*
35.7 ± 0.58
34 ± 1.73
35 ± 2.64
27.7 ± 0.58*
Mutations detected in the genes involved in melanin biosynthesis for A. fumigatus isolates IHEM 2508, 9860 and 15998
Point mutations in genesa
Evidence for conidiation and visualisation of the conidial surface by scanning electron microscopy
Flow cytometry analysis of laminin and fibronectin binding
Flow cytometry analysis of the binding of laminin and fibronectin
Strain or isolate number
Evaluation of the physical properties of the conidial surface
Physical properties of the conidial surface
Strain or isolate number
Zeta potential (mV)
Ultrastructure of the conidial wall visualised by transmission electron microscopy
Visualisation of the hydrophobic rodlet layer by atomic force microscopy
Many fungal species produce pigments such as melanin, either from L-3,4-dihydroxyphenylalanine (the DOPA-melanin pathway, which is more frequently encountered in Basidiomycetes) or from 1,8-dihydroxynaphthalene (the DHN-melanin pathway, usually found in Ascomycetes and relative Deuteromycetes) . The genes and enzymes involved in these metabolic pathways have been known for many years, but the two types of melanin were only recently related to virulence in phytopathogenic or human pathogenic fungi [12–14]. For example, DHN-melanin provides the rigidity of appressoria, which allow the fungus to penetrate plant leaves, in Magnaporthe grisea, the agent responsible for rice blast , and in Colletotrichum lagenarium, responsible for cucurbits disease . The role of melanin in virulence is less well defined in human pathogens such as Cryptococcus neoformans , Paracoccidioides brasiliensis , Exophiala dermatitidis  and Sporothrix schenckii . It has been demonstrated that this pigment protects the fungal cells especially from reactive oxygen species produced by the host immune defences. Brakhage  and Kwon-Chung  demonstrated the importance of melanin for A. fumigatus. They generated white mutants either by UV mutagenesis, or by targeted mutagenesis. These mutants produced white colonies and had mutations in the PKSP (= ALB1) gene, encoding a polyketide synthase required for conidial pigmentation. They were less virulent than their parent wild-type strains in murine models of disseminated aspergillosis, probably due to an increased susceptibility of their conidia to phagocytosis and reactive oxygen species. However, virulence in mice was not affected by the disruption of the ABR2 gene which is involved in a later step of the melanin pathway .
Mutation in the PKSP (ALB1) gene also led to morphological changes of the conidia. Indeed, SEM showed that these pigmentless mutants produced smooth-walled conidia, whereas the conidia of A. fumigatus have typically a rough surface covered with echinulations . The study of mutant isolates of clinical or environmental origin, with defective melanin biosynthesis pathways, suggests that the pigment also plays an indirect role in virulence of A. fumigatus.
Sequencing of the genes involved in the melanin biosynthesis pathway showed a genetic defect in the early steps of this pathway for our isolates. This was consistent with the changes in colony colour observed for reference strains grown in the presence of specific DHN-melanin inhibitors. Two distinct mutations in the ALB1 gene were detected for IHEM 2508 and 9860 isolates, leading to the production of white powdery colonies; whereas the genetic defect was localised in the ARP2 gene for isolate IHEM 15998, producing brown, powdery colonies. As expected, SEM examination of conidial suspensions from our pigmentless isolates showed a smooth surface. However, a lack of ornamentation was also observed on the conidial surface for the brownish isolate, as well as in reference strains cultivated in the presence of pyroquilon, an inhibitor of the hydroxynaphtalene reductase.
Results from flow cytometry experiments confirmed previous work which suggested that the laminin receptors were located on the ornamentations of the conidial wall. Scanning or transmission electron microscopy, showed that labelling was associated mainly with protrusions of the cell wall [21, 22]. The marked decrease in laminin binding receptors to the surface of conidia of mutant isolates compared to reference strains, together with the smooth-walled appearance of these conidia, strengthens our previous conclusions. Previous work  also suggested the presence of at least two distinct adherence systems on the conidial surface in A. fumigatus: 1) the recognition of fibronectin from its tripeptide sequence Arg-Gly-Asp by two fungal polypeptides of 23 and 30 kDa, and 2) the binding of laminin and fibrinogen by a 72-kDa sialic acid-specific lectin located on the ornamentations of the conidial wall . Our current results also support this hypothesis, showing a slight increase in the fibronectin binding capacity of mutant isolates compared with reference strains, together with a marked decrease in the binding of laminin to the conidial surface.
The physical properties of the surface of the conidia were also investigated, as they may contribute to host tissue adherence by bringing interacting surfaces closer and mediating their dehydration. We showed that blockage of the melanin biosynthesis pathway resulted in a marked decrease in the electronegative charge of the conidia, a charge which may be due to ionization of free amine and carboxylic acid groups of some surface proteins. A marked decrease in CSH was also observed for conidia of mutant isolates when compared to reference strains, which was consistent with the increased wettability of the colonies. This result suggests that blockage of the melanin pathway also led to the lack of some hydrophobic components on the conidial surface. The defect in melanin in A. fumigatus mutant isolates could also contribute to the marked loss of adherence properties of their conidia , as melanins are hydrophobic molecules and have a negative charge. Youngchim et al.  localised melanin in the electron dense outer layer of the cell wall which surrounds the conidia by TEM examination of A. fumigatus conidia before and after treatment with enzymes and hot acid. Nevertheless, the precise physico-chemical nature of melanin is not well defined and relationships between melanin and other components of the conidial wall, particularly polysaccharides, remain to be clarified [25, 26].
Among the components of the conidial wall are small proteins called hydrophobins which have been described in a large variety of filamentous fungi including A. fumigatus . Hydrophobins share some common properties. These moderately hydrophobic proteins are secreted into the environment by the fungus and they remain in a soluble form when the fungus is cultivated in a liquid medium. However, at an air-liquid interface (e.g. when the fungus is grown on a solid medium), they assemble in about 10-nm thick rodlets organised in bundles or fascicles on the conidial surface, forming a hydrophobic rodlet layer which may be visualised by AFM.AFM examination of the conidial surface showed that this rodlet layer was lacking in mutant isolates whereas typical rodlets were seen on conidia of the tested reference strain. Immunofluorescence or flow cytometry using specific anti-hydrophobin antibodies should be performed to determine whether or not hydrophobins are totally lacking at the conidial surface or simply not organised into a rodlet layer.
Conidia of A. fumigatus may germinate on contact with water. Previous studies showed major changes in the ultrastructure of the conidial wall during the first stage (swelling) of germination. In addition to a marked increase in cell size and the vacuolisation of the cytoplasm, TEM examination of swollen conidia showed changes in the cell wall which became thinner, probably due to the progressive detachment of the outermost cell wall layer . Conidia of mutant isolates and of reference strains were also examined by SEM and AFM using laminin-coated glass coverslips applied to the centre of sporulating cultures. These experiments confirmed the smooth surface of the conidia of mutant isolates and showed the lack of rodlets at their surface. However, this study was conducted on clinical or environmental isolates with defective DHN-melanin pathways and no isogenic wild-type isolates were available as controls, so other mutations, besides those identified in the melanin pathway may have been responsible for phenotypic changes other than colony colour. Nevertheless, the role of melanin in the organisation of the conidial wall was established, because cultivation of reference strains in a medium containing DHN-inhibitors including pyroquilon led to smooth-walled conidia devoid of the outermost electron-dense layer.
These results demonstrated that, as suggested by Franzen et al. for Fonsecaea pedrosoi , melanin is required for correct assembly of the different layers of the conidial wall in A. fumigatus and, therefore, for the expression of adhesins and other virulence factors at the conidial surface. Due to the complete lack of laminin binding at the surface of their conidia, these pigmentless isolates may be valuable tools in the characterisation of fungal receptors. Comparative studies of the proteins of these isolates and of reference strains are now being undertaken using 2D-electrophoresis.
Unless otherwise specified, all experiments were conducted on three Aspergillus fumigatus isolates from the IHEM Culture Collection (Table 1) producing white (IHEM 2508, IHEM 9860) or brown (IHEM 15998) powdery colonies (Figure 2). Properties of these isolates were compared to those of the reference strain IHEM 18963 (Af293) previously used for genome sequencing of A. fumigatus. Likewise, strain CBS 113.26 previously used in our laboratory for studies on adherence mechanisms in A. fumigatus [9, 21, 30] was also included in these experiments. Both reference strains produced typical, dark blue-green powdery colonies.
Media, growth conditions and preparation of conidial suspensions
Isolates were maintained by weekly passages on yeast extract-peptone-dextrose-agar (YPDA) plates containing in g/L: yeast extract, 5; peptone, 10; glucose, 20; and agar, 20. For some experiments, the organisms were also cultivated on Czapek agar (FeSO4, 7 H2O, 0.01 g; saccharose, 30 g; MgSO4, 0.5 g; KCl, 0.5 g; K2HPO4, 1 g; NaNO3, 3 g; agar, 20 g). Unless otherwise specified, all culture media were supplemented with chloramphenicol 0.5% and cultures were incubated at 37°C for 5 days. Conidia were harvested from 5-day-old cultures on YPDA plates, by scrapping off the mycelium in sterile distilled water, followed by filtration through 28-μm-pore-size nylon filters to eliminate pieces of agar, hyphal fragments and conidial heads. Cells were then pelleted by centrifugation (5 min at 1500 g), washed in sterile distilled water and finally counted using a haemocytometer.
Effect of DHN-melanin inhibitors
Tricyclazole, pyroquilon and fenoxanil (Sigma-Aldrich) were diluted in ethanol and added to Czapek agar, at a final concentration of 20 μg/mL, according to the method of Cunha et al. . Fungal suspensions were prepared as previously described from 5-day-old cultures. After 90 minutes decantation, 50 μL of the supernatant were applied to the surface of the agar plates. Cultures were incubated for 3 days at 37°C. Experiments were conducted in triplicate. Growth controls in Czapek agar without inhibitor and supplemented or not with ethanol, were included for each strain. Statistical analysis was applied, using the unpaired Student's t- test.
DNA extraction and gene sequencing
Oligonucleotides used for gene sequencing
Gene name (gene product)
Genbank accesion no.
Nucleotide sequence (5'-3')
AfALB1 (polyketide synthase)
AfAYG1 (polyketide shortening)
AfARP1 (scytalone dehydratase)
AfARP2 (hydroxynaphtalene reductase)
AfABR1 (vermelone dehydratase)
Nucleotide sequence accession number
ALB1, AYG1, ARP1, ARP2, ABR1 and ABR2 gene sequences determined for strains or isolates CBS 113.26, IHEM 18963, IHEM 2508, IHEM 9860 and IHEM 15998 were deposited in the Genbank database and are available under accession numbers FJ406463 to FJ406498 (see Table 2).
Scanning electron microscopy
Cultures grown through dialysis membranes, conidial suspensions, and conidia fixed on laminin-coated glass coverslips, were examined by SEM. Conidial suspensions were prepared as previously described. For the observation of conidial heads, cultures were grown on YPDA plates through sterile dialysis membranes. After 24 hours incubation, the membrane was removed from the agar plate and then cut into squares (0.5 cm × 0.5 cm) at the periphery of the colony. Round glass coverslips (12 mm diameter) were coated with 500 μL of a laminin solution (10 μg/mL final concentration) in phosphate buffered saline 0.15 M pH 7.2 (PBS) supplemented with 10 mM ethylene-diamine-tetraacetic acid (EDTA) to prevent polymerization of laminin. After 30 min incubation at 37°C under constant shaking, coverslips were washed in PBS. They were then directly applied to the surface of sporulating cultures, and finally washed to remove non adherent conidia.
All samples were fixed with a mix of 2% glutaraldehyde and 2% paraformaldehyde in phosphate buffer 0.1 M under vacuum for 24 hours. After washing, the cells were post-fixed with 2% osmium tetroxyde, then dehydrated by passage through ethanol solutions of increasing concentration (50 to 100%). Finally, ethanol was replaced with hexamethyldisilazane (HMDS) and samples were coated with carbon. Observations were made on a JSM 6301F scanning electron microscope (Jeol, Paris, France) operating at 3 kV and equipped with digital imaging.
Flow cytometry analysis
Human plasma fibronectin and laminin from the murine Englebreth-Holm-Swarm sarcoma tumour (Sigma-Aldrich) were labelled with 5-fluorescein isothiocyanate (FITC; Sigma-Aldrich) by a procedure adapted from Clark and Shepard , as previously described . Binding of laminin and fibronectin to the conidia was analysed by flow cytometry as described previously for A. fumigatus . In these assays, 107 conidia were incubated for 30 min at 37°C under constant shaking with 250 μL of FITC-conjugated protein solution (50 μg/mL final concentration). The cells were then washed, pelleted by centrifugation (3 min at 3500 g) and fixed with 1% formaldehyde in PBS. Experiments were performed in PBS (supplemented with 10 mM EDTA for laminin binding assays). Specificity of the binding was assessed by incubating the cells with the fluorescent laminin or fibronectin in the presence of a 10-fold excess of the same unlabeled protein. All experiments were carried out at least twice and included a negative control performed by incubating the cells with no ligand to ascertain the absence of autofluorescence. Cell surface fluorescence was quantified with a FacsCanto II flow cytometer (Becton-Dickinson). The significance of the difference between two fluorescence frequency distribution histograms (number of fungal cells versus relative fluorescence intensity expressed as arbitrary units on a logarithmic scale) was confirmed by statistical analysis using the Kolmogorov-Smirnoff two sample test. The data presented correspond to mean values of the cell surface fluorescence calculated, in all experiments, from the analysis of about 10,000 cells per sample.
The net surface charge of the conidia was evaluated with a Zetasizer (Malvern Instruments, Worcestershire, United Kingdom) as described by Uyen et al. , by measuring the electrophoretic mobility of the cells in suspension in distilled water (107 conidia/mL). Data were collected from 5,000 cells, and the zeta potential was calculated for each strain using the Helmotz-Smoluchowski equation.
The cell surface hydrophobicity (CSH) was first determined by two-phase partitioning as described by Kennedy et al.  with hexadecane as the hydrocarbon phase. Five hundred microliters of hexadecane were added to 2.5 mL of the conidial suspension (108/mL) in phosphate buffered saline PBS. After vortexing the suspensions (2 min at 2200 vib/min), the tubes were incubated for 10 min at room temperature to allow the two phases to separate. The absorbance of the aqueous phase was then measured at 630 nm (Dynatech MRX revelation) and compared to that of a control consisting of a conidial suspension treated in the same conditions, but without hexadecane. CSH was also determined using a two-aqueous phase system adapted from Cree et al.  and consisting of a mix 1:1 of a 17.5% dextran 260,000 solution (900 μL) and a 14.26% polyethylene glycol (PEG) 3,350 solution (900 μL) in PBS. Two hundred microliters of the conidial suspension in PBS (107 conidia/mL) were added and the obtained suspensions were gently mixed. The tubes were then incubated for 1 hour at room temperature to allow the two phases to separate. Equal volumes (100 μL) of the upper phase rich in PEG (and therefore considered as hydrophobic) and of the lower phase rich in dextran (and therefore considered as hydrophilic) were then sampled and the absorbance of the two phases measured spectrophotometrically at 630 nm. CSH was expressed as the ratio between the absorbance of the upper phase and that of the lower phase.
Transmission electron microscopy
The ultrastructure of the conidial wall was investigated by TEM using conidial suspensions obtained from 5-day-old cultures on YPDA as described above. Fixation, post-fixation, dehydratation and embedding in Epon were as previously described . Thin sections contrasted with uranyle acetate and lead citrate were examined on a JEM-2010 transmission electron microscope (Jeol, Paris, France).
Atomic force microscopy
Conidia were fixed on round glass coverslips coated with laminin, as previously described for MEB. The samples were washed in PBS buffer and then dried at room temperature before AFM analysis on a Thermomicroscopes Autoprobe CP Research (Veeco Instruments, Sunnyvale, CA, USA).
About the Authors
MP, GT, DC, FS and JPB are members of the ISHAM Working group on Chronic respiratory infections in cystic fibrosis.
This work was supported by a Belgian Science Policy grant (action for the promotion and co-operation with the Belgian Coordinated Collections of Micro-organisms, BCCM; contract C3/00/19).
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