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Activation of Melanin Synthesis in Alternaria infectoria by Antifungal Drugs Chantal Fernandes,a Rafael Prados-Rosales,b,g Branca M. A. Silva,a Antonio Nakouzi-Naranjo,b Mónica Zuzarte,c,f Subhasish Chatterjee,d Ruth E. Stark,d Arturo Casadevall,e Teresa Gonçalvesa,f CNC—Centre for Neurosciences and Cell Biology, University of Coimbra, Coimbra, Portugala; Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, New York, New York, USAb; Centre of Ophthalmology and Vision Sciences, Institute of Biomedical Imaging and Life Sciences (IBILI), Faculty of Medicine, University of Coimbra, Coimbra, Portugalc; Department of Chemistry, City College of New York, Graduate Center, and Institute for Macromolecular Assemblies, City University of New York, New York, New York, USAd; Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, Baltimore, Maryland, USAe; FMUC—Faculty of Medicine, University of Coimbra, Coimbra, Portugalf; CIC bioGUNE, Derio, Bizkaia, Spaing

The importance of Alternaria species fungi to human health ranges from their role as etiological agents of serious infections with poor prognoses in immunosuppressed individuals to their association with respiratory allergic diseases. The present work focuses on Alternaria infectoria, which was used as a model organism of the genus, and was designed to unravel melanin production in response to antifungals. After we characterized the pigment produced by A. infectoria, we studied the dynamics of 1,8dihydroxynaphthalene (DHN)-melanin production during growth, the degree of melanization in response to antifungals, and how melanization affected susceptibility to several classes of therapeutic drugs. We demonstrate that A. infectoria increased melanin deposition in cell walls in response to nikkomycin Z, caspofungin, and itraconazole but not in response to fluconazole or amphotericin B. These results indicate that A. infectoria activates DHN-melanin synthesis in response to certain antifungal drugs, possibly as a protective mechanism against these drugs. Inhibition of DHN-melanin synthesis by pyroquilon resulted in a lower minimum effective concentration (MEC) of caspofungin and enhanced morphological changes (increased hyphal balloon size), characterized by thinner and less organized A. infectoria cell walls. In summary, A. infectoria synthesizes melanin in response to certain antifungal drugs, and its susceptibility is influenced by melanization, suggesting the therapeutic potential of drug combinations that affect melanin synthesis.

T

he nonspecific term “melanin” describes a group of diverse and complex high-molecular-mass polymers that are characterized by a negative charge and hydrophobicity and that have been associated with a wide variety of functions in many different organisms (1). These dark pigments, which are negatively charged and hydrophobic and which have high molecular masses, are produced by animals, plants, and microbes. They are formed by the oxidative polymerization of phenolic or indolic compounds. In fungi, melanins may be synthesized from an endogenous substrate via a 1,8-dihydroxynaphthalene (DHN) intermediate or, alternatively, from L-3,4-dihydroxyphenylalanine (L-DOPA) (2). Other melanins called pyomelanins are derived from L-tyrosine via homogentisic acid (3). Alternaria spp. are saprophytic fungi of the order Pleosporales, which includes fungi of the family Dematiaceae with melanized cell walls (4). The members of this genus are ubiquitous environmental fungal agents associated with human allergies (5) and phaeohyphomycosis, a cutaneous or subcutaneous pathology that can turn into a severe disease in individuals with impaired immunity (6). A growing number of reports also associate sensitivity to Alternaria with asthma (5, 7–9), possibly due to the high spore germination rate of members of this genus and their release of protease (10). The rise in the incidence of asthma among adults and children may be linked to global climate changes and reductions in the ozone layer and the accompanying increased solar radiation (5, 11, 12). Since the melanin of fungal cell walls provides protection against UV, ionizing, and gamma radiation (13), this pigment may contribute to the higher survival rates and competitive abilities of melanized fungi, leading to further environmental proliferation. Additionally, melanins can protect against

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antifungal drugs (13) and the free oxygen radicals generated by host defense mechanisms (1, 14). In Alternaria spp., only the DHN-melanin pathway for melanin synthesis has been described (15–21), and the gene cluster required for melanin synthesis was cloned from Alternaria alternata (15). In Alternaria spp., melanin is present in the conidia and is confined to the outer region of the primary cell walls and the septa, which delimit individual spore cells in the multicellular conidium. After the delimitation of the cells by septa, secondary unmelanized cell walls are deposited (16, 22). This pigment is likely to be involved in conidial development, since it has been shown that disruption of a melanin biosynthesis gene in A. alternata leads to a reduction in the conidial size as well as septal number (18). Although melanin is associated with virulence in several ani-

Received 9 September 2015 Returned for modification 4 October 2015 Accepted 15 December 2015 Accepted manuscript posted online 28 December 2015 Citation Fernandes C, Prados-Rosales R, Silva BMA, Nakouzi-Naranjo A, Zuzarte M, Chatterjee S, Stark RE, Casadevall A, Gonçalves T. 2016. Activation of melanin synthesis in Alternaria infectoria by antifungal drugs. Antimicrob Agents Chemother 60:1646 –1655. doi:10.1128/AAC.02190-15. Address correspondence to Teresa Gonçalves, [email protected]. C.F. and R.P.-R. contributed equally to this article. A.C. and T.G. share senior authorship. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /AAC.02190-15. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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mal-pathogenic fungi, like Aspergillus fumigatus or Cryptococcus neoformans (23–28), no such association has been made for A. alternata or Alternaria brassicicola. Since Alternaria spp. naturally produce unmelanized appressoria, it is not surprising that A. alternata and A. brassicicola mutants that are deficient in melanin production retain their pathogenicity toward their hosts, which are usually plants (19, 20, 29). In the present work, we aimed to study the impact of Alternaria infectoria melanin on the salvage mechanisms of the response to antifungals and explored whether melanin synthesis inhibition in combination with antifungal drugs acts synergistically to efficiently fight fungi with melanized cell walls. We found that A. infectoria produces DHN-melanin, which is deposited in the outermost layer of the hyphal or conidial cell wall of fungi, and that only pyroquilon, a DHN-melanin synthesis inhibitor, and not glyphosate, a DOPA-melanin inhibitor, arrests the synthesis of the dark pigment. We also report that pyroquilon inhibits A. infectoria mycelial growth, while exposure to the antifungals caspofungin, nikkomycin Z, and itraconazole but not fluconazole or amphotericin B stimulates the synthesis of melanin. Furthermore, we found that the combination of pyroquilon with caspofungin leads to a decrease in the minimum effective concentration (MEC) of caspofungin. The latter combination also resulted in more swollen hyphal balloons and thinner cell walls. MATERIALS AND METHODS Organisms and media. A. infectoria strain CBS 137.90 was purchased from the Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Center. Another A. infectoria clinical isolate was used for determination of the polyketide synthase nucleotide sequence. The latter isolate was previously obtained by us (30) and is deposited in the CBS collection as strain CBS 122351. A. alternata was kindly provided by R. Freitas and R. M. B. Ferreira (Instituto Superior de Agronomia, Universidade de Lisboa, Lisbon, Portugal). Fungi were stored at ⫺80°C. Cultures were grown on potato dextrose agar (PDA; Difco) or yeast-malt extract (YME; 4 mg/ml yeast extract, 10 mg/ml malt extract, 10 mg/ml dextrose). Nikkomycin Z, pyroquilon (1,2,5,6-tetrahydropyrrolo [3,2,1,-ij]quinolin-4one), amphotericin B, and glyphosate were purchased from SigmaAldrich (St. Louis, MO), and caspofungin was a gift from Merck & Co, Inc., Rahway, NJ (material transfer agreement no. 37006). Fluconazole was obtained from Pfizer, and itraconazole was purchased from Actavis as 100-mg capsules (purity, 25%; excipient, sugar spheres [sucrose and maize starch], poloxamer, and hypromellose). Fluconazole, itraconazole, and amphotericin B were suspended in dimethyl sulfoxide (DMSO) to achieve stock solutions with concentrations of 38 mg/ml, 5 mg/ml, and 10 mg/ml, respectively. Preparation of inoculum and growth conditions. We previously described the procedure used here to prepare the inocula and the growth conditions (31). Briefly, spores of A. infectoria were collected from 2-week-old PDA plate cultures that had been grown at 28°C with an alternating 8-h light and 16-h dark (day-night) cycle under an F15W T8BLB lamp. The mycelium was submerged in liquid YME medium and scraped with an inoculation loop, and the homogenate was used as the inoculum. Liquid A. infectoria cultures were cultivated on YME with or without drug supplementation at 28°C with constant orbital shaking at 180 rpm and by use of the day-night cycle described above. Fungal growth for characterization of the accumulated pigments. YME (200 ml) with or without 25 ␮g/ml of pyroquilon was inoculated with 5 ⫻ 106 conidia of either A. infectoria or A. alternata, and the conidia were incubated for 4 days on a rotary shaker at 120 rpm at 28°C and by use of the day-night cycle described above. The mycelial mats were harvested by filtration in a steel filter, washed 4 times with distilled water, and frozen at ⫺20°C until melanin extraction.

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Melanin synthesis from A. infectoria cultures. Several Erlenmeyer flasks containing 50 ml of YME were inoculated with 1 ⫻ 106 A. infectoria conidia and incubated on a rotary shaker at 120 rpm and 28°C under an alternating day-night cycle of 8 h of light enriched with UV and 16 h of dark. The cultures were analyzed every 24 h for 11 days for pigment formation and dry weight determination to assess fungal growth. For quantification of the accumulation of melanin in the fungal cell wall in response to antifungals, Erlenmeyer flasks containing 50 ml of YME supplemented with itraconazole (0.08 and 0.25 ␮g/ml), fluconazole (8 and 16 ␮g/ml), caspofungin (1 and 2 ␮g/ml), nikkomycin (0.5 and 5 ␮g/ml), amphotericin B (1 and 4 ␮g/ml), and DMSO (0.02%) were inoculated and incubated under the conditions described above for 4 days. The mycelial mats were harvested by filtration in a steel filter, washed 4 times with distilled water, and frozen at ⫺20°C until melanin extraction. To estimate fungal biomass, the material was freeze-dried and weighed. MIC and MEC determination in the presence of pyroquilon. MICs and MECs were determined according to the standardized protocol (the M38A protocol) for filamentous fungi developed by the Clinical and Laboratory Standards Institute (CLSI). Briefly, A. infectoria spores were suspended in sterile normal saline and inoculated in RPMI 1640 medium with L-glutamine (catalog number R8758; Sigma). Final drug concentrations ranged from 0.1 to 15 ␮g/ml for amphotericin B, 0.5 to 2.0 ␮g/ml for caspofungin, 0.01 to 1.0 ␮g/ml for nikkomycin Z and itraconazole, and 4 to 50 ␮g/ml for fluconazole in the presence or absence of pyroquilon at 25 ␮g/ml for all antifungals. Pyroquilon is a specific inhibitor of the DHNmelanin biosynthesis that interferes with the dehydrogenation of 1,3,6,8tetrahydroxynaphthalene (1,3,6,8-THN) to scytalone and 1,3,8-trihydroxynaphthalene (1,3,8-THN) to vermelone (32). As previously described (31), the MECs of caspofungin and nikkomycin Z were defined to be the lowest concentrations resulting in aberrant hyphal growth (33). The MECs were determined at 72 h, and the hyphae were visualized at 120 h under a Nikon Eclipse E400 microscope equipped with a digital camera (Nikon Digital Sight DS-L1). Itraconazole and fluconazole MIC values were the lowest drug concentrations preventing visible growth in a broth dilution susceptibility test and were assessed after incubation at 35°C for 72 h. Pigment isolation from mycelia. For extraction of melanin from A. alternata and A. infectoria mycelia or conidia (inoculum), the freeze-dried mycelial mats and conidia were suspended in 1 M NaOH and ground with a mortar and pestle. The ground material was then extracted with 1 M NaOH and autoclaved at 121°C for 30 min. 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). Melanin ghost preparation. A. infectoria melanin ghosts, i.e., particles obtained by chemical and enzymatic removal of the other cellular structures (37), were prepared for measurement of the zeta potential, scanning electron microscopy (SEM), determination of the electron spin resonance (EPR) spectrum, and measurement of the nuclear magnetic resonance (NMR). These were prepared either from conidia or from hyphae, as described previously (38). Briefly, fungal structures were collected by centrifugation at 4,000 rpm, and the pellet was suspended in 1 M sorbitol– 0.1 M sodium citrate solution. Cell wall-lysing enzymes from Trichoderma harzianum (Sigma-Aldrich, St. Louis, MO, USA) were added at a final concentration of 10 mg/ml, and the suspension was incubated at 30°C for 24 h. Protoplasts were collected by centrifugation and incubated with 4 M guanidinium thiocyanate for 12 h with gentle shaking at room temperature. The remaining material was collected by centrifugation, suspended in 1 mg/ml of proteinase K in reaction buffer (10 mM Tris, pH 7.8, 1 mM CaCl2·2H2O, 0.5% SDS), and incubated at 65°C for 4 h. The particles were then collected by centrifugation, washed in phosphate-buffered saline

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FIG 1 A. infectoria melanin synthesis during germination and mycelial growth. (A) Quantification of the melanin content in A. infectoria cells over 11 days in liquid cultures. Bars, mycelium dry weight; diamonds, melanin content, measured by determination of the absorbance at 340 nm, as described in Materials and Methods, and given as the absorbance (Abs) per milligram (dry weight) of mycelia. At the first stage of growth, the high melanin contents are likely due to the melanin present in the germinating melanized spore that remained at the center of the mycelium. (B) Macroscopic view of mycelia collected from liquid cultures of A. infectoria over the 11 days of growth (panels 1 to 11, respectively.).

(PBS), suspended in 6 M HCl, boiled for 1 h, and finally, dialyzed against water for 7 days and lyophilized. Growth inhibition by pyroquilon and glyphosate. The inhibition of A. infectoria mycelial growth by pyroquilon and glyphosate was estimated by an agar plate method. The autoclaved PDA medium was maintained in a water bath at 45°C, and either glyphosate or pyroquilon was added to the following final concentration: 10, 25, 50, or 100 ␮g/ml. The homogeneous mixtures were poured into sterilized petri dishes. The center of these dishes was inoculated with 5 ␮l of a suspension of 2.5 ⫻ 106 spores/ml. Plates containing spore suspensions without glyphosate or pyroquilon were used as negative controls. All plates were incubated at 28°C for at least 8 days by use of the day-night cycle described above. Fungal growth was measured as the colony diameter and is expressed in terms of the percent inhibition of mycelia using the following formula: [(Dc ⫺ Dt)/Dc] · 100, where Dc is the diameter (in millimeters) of the control colony and Dt (in millimeters) is the diameter of the treated colony. SEM study. For SEM, samples were fixed with 2.5% glutaraldehyde, 0.1 M sodium cacodylate, 0.2 M sucrose, and 5 mM MgCl2 at pH 7.4 and dehydrated through a graded series of ethanol. Drying to the critical point was accomplished using liquid carbon dioxide in a Toumisis Samdri 795 critical point dryer (Rockville, MD, USA). Sputter coating was done with gold-palladium in a Denton Vacuum Desk-2 sputter coater (Cherry Hill, NJ, USA). Samples were examined in a Zeiss Supra field emission scanning electron microscope (Carl Zeiss Microscopy, LLC, North America), using an accelerating voltage of 5 kV. TEM. Transmission electron microscopy (TEM) analysis was performed in two sets of experiments. First, we studied the melanization of the spores. Samples of spore suspensions were fixed with 4.0% paraformaldehyde and 5% glutaraldehyde in 2⫻ PBS buffer mixed 1:1 with growth medium, enrobed in 4% gelatin, postfixed with 1% osmium tetroxide followed by 1% uranyl acetate, dehydrated through a graded series of ethanol, and embedded in Spurr resin (Electron Microscopy Sciences, Hatfield, PA). Ultrathin sections were cut on a Reichert Ultracut UCT microtome, stained with uranyl acetate followed by lead citrate, and viewed in a JEOL 1200EX transmission electron microscope at 80 kV. In a second set of experiments, we studied the changes to the cell wall due to antifungal and pyroquilon exposure. Liquid A. infectoria cultures were cultivated on YME with or without drug supplementation (1.5 ␮g/ml of caspofungin, 25 ␮g/ml of pyroquilon, 1.5 ␮g/ml of caspofungin plus 25 ␮g/ml of pyroquilon, or 0.08 ␮g/ml of nikkomycin Z) at 28°C with constant orbital shaking at 180 rpm for 4 days with the day-night cycles described above. The samples were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.2) for 2 h. Postfixation was performed using 1% osmium tetroxide for 1 h. After they were rinsed with the buffer,

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the samples were dehydrated in a graded ethanol series (30 to 100%), impregnated, and embedded in Epoxy resin (Fluka Analytical). Ultrathin sections (⬃80 nm) were mounted on copper grids (300 mesh) and stained with uranyl acetate 2% (15 min) and 0.2% lead citrate (10 min). Observations were carried out on an FEI-Tecnai G2 Spirit Bio Twin transmission electron microscope at 100 kV. Statistical analysis. Data were analyzed by Student’s t test using Prism (version 5) software (GraphPad Software, Inc., La Jolla, CA). Data are presented as the means ⫾ standard errors of the means (SEMs) or standard deviations (SDs), and differences were considered significant at P values of ⬍0.05.

RESULTS

Melanin pigment production during A. infectoria growth. A. infectoria spores collected from PDA plates manifested extensive melanization. After their germination and growth in YME medium for 11 days, we observed that the A. infectoria melanin contents increased along the growth (Fig. 1). The presence of a DHNmelanin was confirmed by the strong UV absorbance, zeta potential values, and Fourier transform infrared spectroscopy (FTIR), solid-state 13C NMR, and EPR spectra typical of fungal melanins (see the supplemental material). The accumulation of melanin in the A. infectoria mycelium was quantified photometrically at 340 nm as described in the Materials and Methods. We initially observed high levels of melanin to be associated with the spores. The melanin content dropped during the initial germination, but at day 4 melanin accumulation resumed and the melanin content at day 11 was half of that of the spores (Fig. 1A). This increase in melanin content was also visible macroscopically (Fig. 1B). We conducted a comparative study with A. alternata, concluding that while A. alternata releases melanin to the growth medium, A. infectoria does not (results not shown). Melanin ghosts isolated from A. infectoria conidia vary in size and structural morphology. A. infectoria conidia manifest an external electron-dense layer in transmission electron micrographs (Fig. 2A). The same technique showed that in melanin ghosts obtained from conidia, most of the electron-dense material is restricted to the outer layer of the conidial cell wall (Fig. 2B). SEM analysis showed large variations in particle size, including globular structures protruding from the main body, with the sizes of those structures ranging from 1 to 10 ␮m (Fig. 2C). Scanning electron micrographs of A. infectoria melanin from conidia

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FIG 2 A. infectoria melanin localization. (A) Transmission electronic micrograph of typical A. infectoria conidia showing an electron-dense surface and a less electron-dense compartment corresponding to a germinating spore (arrow); (B) TEM of a melanin ghost particle; (C) scanning electron micrographs of A. infectoria conidia; (D) scanning electron micrographs of A. infectoria melanin isolated from conidia. Bars, 1 mm (C, top) and 200 nm (C, bottom, and D).

showed a partially destroyed conidial structure, including many tightly aggregated clusters of spherical granules with an average diameter of 100 to 300 nm (Fig. 2D). Higher-magnification images revealed the inside of the aggregated structure and clearly showed that the surfaces had multisized stacks of particles. Melanization in the presence of melanin inhibitors. To study the efficiency of melanin inhibitors in A. infectoria melanization, we used pyroquilon, a specific inhibitor of DHN-melanin biosynthesis. The mycelial mat changed from black to a reddish brown color, and a typically reddish pigment surrounding the colony indicated that melanin biosynthesis was inhibited (Fig. 3). The inhibition of A. infectoria radial growth was also quantified to evaluate if pyroquilon interfered with the further determination of the MECs of the antifungals. With 25 ␮g/ml of pyroquilon, melanization was inhibited, while the inhibition of growth was only 10% (Fig. 3). The percentage of radial growth inhibition doubled in the presence of 50 ␮g/ml pyroquilon (Fig. 3), but a concentration of 100 ␮g/ml of pyroquilon hardly increased the percentage of radial growth inhibition (from 20.2% ⫾ 5.0% to 22.5% ⫾ 4.7%). We also tested glyphosate, a DOPA-melanin inhibitor, but the mycelial mat of A. infectoria remained black, and this compound did not inhibit the radial growth, indicating that the L-DOPA

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pathway is not responsible for melanization in these fungi (data not shown). Melanization and susceptibility to antifungal drugs. Since melanin is a component of the cell wall of dematiaceous fungi, we studied in A. infectoria how melanization was influenced by the presence of diverse antifungals, such as cell wall synthesis inhibitors (caspofungin and nikkomycin Z), ergosterol synthesis inhibitors (itraconazole and fluconazole), and a polyene, an ergosterolbinding drug (amphotericin B). Furthermore, we aimed to unravel if the lack of melanin influenced the antifungal efficiency of caspofungin, nikkomycin Z, itraconazole, fluconazole, and amphotericin B. The assays were performed with 25 ␮g/ml pyroquilon, since at that concentration the melanization of A. infectoria was inhibited but the fungal growth decreased only 10% (Fig. 3) and no morphological alterations were observed. A. infectoria responded with increased melanization when it was grown in the presence of caspofungin, nikkomycin Z, and itraconazole but not in the presence of fluconazole (Fig. 4). A slight increase in melanization was observed with amphotericin B, but it was not statistically significant (Fig. 4). The MIC values of itraconazole (0.1 ␮g/ml) and fluconazole (10 ␮g/ml) obtained were lower than those described in the literature for Alternaria spp., being 0.5 ␮g/ml and 32 ␮g/ml, respectively (39). The am-

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FIG 3 Impact of melanin synthesis inhibition on the growth of A. infectoria. (A to D) An A. infectoria conidial suspension was inoculated in the center of PDA plates. The radial growth of A. infectoria was measured in the absence of pyroquilon (A) and presence of 10, 25, and 50 ␮g/ml of pyroquilon (B to D, respectively). Observation of the plates also allows the visualization of the changes in fungal melanization and the accumulation of a reddish pigment. (Right) Quantification of the inhibitory effect of several concentrations of pyroquilon on the radial growth of A. infectoria. The results reported are the means ⫾ SDs from 3 independent experiments. The radial growth diameter varied from 4.2 to 4.3 cm for PDA, 3.7 to 4.0 cm in PDA plus 10 ␮g/ml of pyroquilon, 3.7 to 4.0 cm in PDA plus 25 ␮g/ml of pyroquilon, and 3.2 to 3.7 cm in PDA plus 50 ␮g/ml of pyroquilon.

photericin B MIC was 1.3 ␮g/ml, in agreement with a previous report (39). Nevertheless, these values remained unchanged in the presence of 25 ␮g/ml pyroquilon. For caspofungin and nikkomycin Z, we used a distinctive parameter, the minimum effective concentration (MEC). MEC defines the lowest concentration of the drug yielding morphological alterations, such as those observed with caspofungin, which exerts antifungal activity at actively growing tips and the branching points of Aspergillus hyphae, leading to the formation of flattening and swelling tips (33). In A. infectoria, caspofungin and nikkomycin Z lead to the formation of abnormal balloon-like cells (31, 40). We measured a nikkomycin Z MEC of 0.5 ␮g/ml, since this was the minimal concentration required for the occurrence of hyphae with balloon-like features. The presence of pyroquilon did not decrease this MEC. In contrast, the simultaneous presence of 25 ␮g/ml pyroquilon and

FIG 4 Melanin accumulation in A. infectoria in response to itraconazole, fluconazole, nikkomycin Z, caspofungin, and amphotericin B at subinhibitory and inhibitory concentrations. The melanin content is normalized to the fungal biomass and expressed as a percentage of the melanin content of the control. The results reported are the means ⫾ SEMs from 3 independent experiments. *, P ⬍ 0.05; **, P ⬍ 0.01; ***, P ⬍ 0.001. Ctrl, control; Nikko. Z, nikkomycin Z; Amphot. B, amphotericin B.

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caspofungin led to a decrease in the MEC value from 1.4 ␮g/ml to 1.0 ␮g/ml. Furthermore, some of the balloon-shaped hyphae that occurred when pyroquilon was associated with caspofungin were larger (20.4 ⫾ 0.9 ␮m in height by 20.0 ⫾ 0.8 ␮m in width) than those seen with caspofungin alone (13.2 ⫾ 0.6 ␮m in height by 12.2 ⫾ 0.6 ␮m in width) (Fig. 5). To evaluate the impact of caspofungin, pyroquilon, and nikkomycin Z alone and the combination of caspofungin and pyroquilon on the A. infectoria cell wall, we acquired TEM micrographs

FIG 5 Effect of the combination of caspofungin and pyroquilon (Pyr) on the A. infectoria hyphal structure. Arrows, hyphal balloons of larger size resulting from the presence of pyroquilon. Bars, 30 ␮m.

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FIG 6 Transmission electron microscopic observations of the A. infectoria hyphal cell wall morphology and thickness in the absence or presence of caspofungin, pyroquilon, caspofungin plus pyroquilon, and nikkomycin Z. The fungus was grown in YME (control) and in YME with 1.5 ␮g/ml of caspofungin (Cas), 25 ␮g/ml of pyroquilon (Pyr), 1.5 ␮g/ml of caspofungin plus 25 ␮g/ml of pyroquilon, or 0.08 ␮g/ml of nikkomycin Z. The results of the cell wall thickness measurement reflect the means ⫾ SEMs of at least 20 random measurements. **, P ⬍ 0.01; ***, P ⬍ 0.001. Arrow a, extracellular vesicle; arrow b, Woronin body.

of fungal cells treated and not treated with antifungals (Fig. 6). Untreated A. infectoria cells showed a well-defined surface coating of electron-dense material (outermost layer) adjacent to the outer cell wall layer. The inner cell wall and the plasma membrane also had regular structures. In contrast, caspofungin, pyroquilon, caspofungin plus pyroquilon, or nikkomycin Z treatment led to alterations in the organization of the cell wall structures. The inner

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and outermost layers of the plasma membrane were irregular. Upon caspofungin treatment, we observed abnormalities of the septal structures of the cells compared to the appearance of the septal structures under the control conditions (Fig. 6). In fact, septal pores were seen, together with the plugging of Woronin bodies (Fig. 6), indicating that caspofungin probably also affected the septal structure. The combination of caspofungin and pyro-

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quilon led to a particularly high level of disorganization of the outer electron-dense cell wall, with loose fragments being seen on the surface of the cell wall (Fig. 6). Nikkomycin Z treatment also caused a severe change to the organization of the cell wall structure, which became irregular, with loose material being seen at the cell wall surface. We also observed a decrease in the cell wall thickness with the caspofungin and caspofungin plus pyroquilon treatments in relation to the thickness of the control cells (Fig. 6). DISCUSSION

Melanin is usually associated with a variety of cellular protective effects in biological systems, including increased resistance to UV radiation, free radicals, and antifungals. In the present work, we report that A. infectoria, a fungal species belonging to the Dematiaceae, produces DHN-melanin, which accumulates in the cell wall during fungal growth. A. infectoria increases melanin synthesis in response to nikkomycin Z, caspofungin, and itraconazole and less extensively in response to amphotericin B and fluconazole. To infer whether this increased melanization is a defense mechanism against antifungals and to evaluate whether there exists a hypothetical synergistic effect between antifungals and melanin synthesis inhibitors, similar to our previous findings with a combination of antifungals targeting chitin and ␤-glucan from the cell wall (40), we determined the MECs and MICs of caspofungin, nikkomycin Z, itraconazole, fluconazole, and amphotericin B in the presence and absence of pyroquilon. We found a decrease in the MEC of caspofungin and an increase in the numbers of hyphal balloons when A. infectoria was grown in the presence of pyroquilon and caspofungin. These findings could reflect a cell wall lacking both ␤-glucan and melanin. In addition, we observed a decrease in cell wall thickness when caspofungin was combined with pyroquilon. A. infectoria spores collected from PDA medium manifested extensive melanization in comparison with the level of melanization in the mycelial mat. This finding is consistent with previous data showing that melanin is probably actively involved in conidial development, since disruption of a melanin biosynthesis gene in A. alternata reduced the conidial size as well as the septal number (18). Moreover, we demonstrated that during conidial germination the melanin content decreased, but it increased with mycelial growth and maturation. In fact, TEM images showed that when germination was initiated, the surface of the emergent structure was less melanized (Fig. 2A). TEM analysis also showed that most of the melanin was located in the cell wall of the conidium, which is a common feature in dematiaceous fungi and other filamentous fungi that produce melanin, such as Aspergillus niger (41). As described in other fungi, such as A. fumigatus (42), we also confirmed the presence of a well-defined surface coating of electron-dense material at the outermost layer of the cell wall of A. infectoria hyphae that was perturbed when the A. infectoria mycelium was exposed to the melanin synthesis inhibitor pyroquilon (Fig. 6). As noted above, the term “melanin” describes a heterogeneous group of pigmented polymers. We performed an extensive study, using elemental analysis, UV/infrared absorption spectrophotometry, measurement of the zeta potential, and solid-state NMR analysis (43–46), in order to identify the chemical characteristics of A. infectoria melanin (see the supplemental material) and found that A. infectoria melanin-derived pigments exhibited substantial absorption in the UV spectrum and none in the visible light spec-

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trum, as well as diverse spectroscopic characteristics shared with other melanins described to date (47), including A. alternata DHN-melanin (see the supplemental material). To ascertain the chemical nature of the melanin, we used specific inhibitors of the DHN-melanin (pyroquilon) and of the DOPA-melanin (glyphosate) synthetic pathways. In vitro, the melanization of A. infectoria can be blocked with an inhibitor of the DHN pathway but not with an inhibitor of the DOPA-pathway. As others have observed (32, 48, 49), the inhibition of A. infectoria DHN-melanin synthesis resulted in the production of a carmine red pigment rather than an albino phenotype. Consequently, we conclude that A. infectoria synthesizes DHN-melanin, which is similar to the melanins described to be synthesized by other Alternaria spp. (15–21). This claim was corroborated by the identification in A. infectoria of the genes encoding enzymes involved in DHN-melanin synthesis (2), trihydroxynaphthalene reductase, polyketide synthase, and scytalone dehydratase (see the supplemental material). Electron microscopy revealed a spherical granular body arrangement in clusters with different sizes and aggregations. Higher magnifications revealed a substructure of spherical units that were variable in size. As in C. neoformans, the melanin is deposited in the cell wall, and after the enzymatic degradation of the cell wall, the melanin structure retained the shape of the cells, resulting in hollow spheres called “ghosts” (2, 50). One of the main objectives of the present study was to unravel putative synergies between antifungals and melanin synthesis inhibitors. Previously, DHN-melanin from Madurella mycetomatis was reported to bind to itraconazole and ketoconazole, resulting in an in vitro decrease in the MIC because of the lack of drug accessibility to the fungus (51). On the other hand, various melanin synthesis inhibitors, such as pyroquilon and tricyclazole, have been used for rice blast control, and since these inhibitors do not exhibit direct activity on fungal growth, there should be a low risk of emergence of tolerant strains (52). We report that in A. infectoria the presence of pyroquilon did not change the MICs of the azoles itraconazole and fluconazole. It was established that for Aspergillus spp. the growth inhibition by itraconazole is 100% (53, 54). However, it is worth mentioning that Aspergillus niger is the Aspergillus species with the lowest susceptibility to itraconazole (53). We now describe that A. infectoria increases the synthesis of melanin in response to itraconazole, raising the question of whether the lower susceptibility of A. niger to itraconazole results from a higher melanin content. One of our most important findings was that pyroquilon increased the antimicrobial efficiency of caspofungin in A. infectoria. This conclusion arose from several observations, including a decrease in the MEC, a decrease in the cell wall thickness, an increase in the sizes of the balloons, and a change in the organization of the cell wall. It was described first by Valentine and Bainbridge (1978) (55) and then by the Douglas group (56) that ballooning corresponds to hyphal fragments that are osmosensitive and so more prone to burst in response to osmotic stress or to other stresses, such as temperature. Since the combination of pyroquilon and caspofungin increases the size of the balloons, we conclude that the synergistic effect between pyroquilon and caspofungin would increase the fungicidal potential of this echinocandin. In fact, caspofungin, which inhibits the synthesis of 1,3-␤-D-glucan, is fungicidal against some yeasts (most species of Candida) but is fungistatic against some molds (Aspergillus but not Fusarium and Alternaria) and modestly or minimally active against dimorphic

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fungi (Blastomyces and Histoplasma) (57, 58). Nevertheless, the action of echinocandins can be improved due to their synergistic effects when they are combined with polyenes or azoles (59). We now report that the inhibition of DHN-melanin can enhance the efficiency of echinocandins. We have previously shown in A. infectoria that while the echinocandin caspofungin or the polyoxin nikkomycin Z (an inhibitor of chitin synthesis) alone provided only fungistatic inhibition, the combination of both led to fungal cell lysis in vitro (31, 40). On the other hand, in contrast to the findings for other fungi, such as A. fumigatus and Candida albicans, A. infectoria strain IMF006 was not provided with the salvage mechanism (40) that leads to an increase in the chitin content of the cell wall in response to inhibition of 1,3-␤-D-glucan synthesis by echinocandins and that is responsible for attenuation of the in vitro activity of caspofungin at higher concentrations, called the paradoxical effect (60–62). The present work suggests that this lack of alteration of chitin levels is compensated for by melanin accumulation, which makes the overall cell wall architecture stiffer, and for that reason, in the presence of pyroquilon, a significant alteration of the morphology of the cell occurs at lower concentrations of caspofungin. 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. 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. 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. Interfering with melanin synthesis using the melanin inhibitor glyphosate can produce a therapeutic effect independent of the action of antifungal agents (65). Agents that interfere with melanin synthesis in A. infectoria could render the fungal cells more vulnerable to the host immune system without affecting the host melanin synthesis pathways, and consequently, this effect is of potential pharmacological interest and is independent of any effect that such agents would have on antifungal drug efficacy. In summary, our observations reinforce the fact that melanin is an integral cell wall component that forms a shielding structure with a significant impact on the overall architecture of the cell wall and show that a combination of agents targeting cell wall synthesis and melanin synthesis produces effects that could potentially be exploited therapeutically.

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ACKNOWLEDGMENTS We thank the Thermodynamics/Solid State Chemistry Laboratory, SER Group, Coimbra Chemistry Center, for FTIR spectroscopy.

FUNDING INFORMATION This study was partly supported by a project funded by the Fundação para a Ciência e Tecnologia (FCT), PTDC/SAU-ESA/108636/2008, cofunded by COMPETE-Operational Programme Competitiveness Factors and FEDER, by FEDER funds through COMPETE, and by national funds provided by FCT under strategic project UID/NEU/04539/2013. C.F. is the recipient of a postdoctoral fellowship from FCT (SFRH/BPD/63733/ 2009). B.M.A.S. was the recipient of a research fellowship within the scope of FCT project PTDC/SAU-ESA/108636/2008. This research was also supported by a grant from the National Institutes of Health (NIH R01AI052733). The 600-MHz NMR facilities used in this work are operated by the City College of New York and the City University of New York Institute for Macromolecular Assemblies, with additional infrastructural support being provided by grant 8G12 MD007603-29 from the National Institute on Minority Health and Health Disparities of the National Institutes of Health.

REFERENCES 1. Langfelder K, Streibel M, Jahn B, Haase G, Brakhage AA. 2003. Biosynthesis of fungal melanins and their importance for human pathogenic fungi. Fungal Genet Biol 38:143–158. http://dx.doi.org/10.1016/S1087 -1845(02)00526-1. 2. Eisenman HC, Casadevall A. 2012. Synthesis and assembly of fungal melanin. Appl Microbiol Biotechnol 93:931–940. http://dx.doi.org/10 .1007/s00253-011-3777-2. 3. Yabuuchi E, Ohyama A. 1972. Characterization of pyomelaninproducing strains of Pseudomonas aeruginosa. Int J Syst Bacteriol 22:53– 64. http://dx.doi.org/10.1099/00207713-22-2-53. 4. de Hoog GS, Guarro J, Gené J, Figueras MJ. 2000. Atlas of clinical fungi, 2nd ed. Centraalbureau voor Schimmelcultures, Utrecht, The Netherlands, and Universitat Rovira i Virgili, Reus, Spain. 5. Knutsen AP, Bush RK, Demain JG, Denning DW, Dixit A, Fairs A, Greenberger PA, Kariuki B, Kita H, Kurup VP, Moss RB, Niven RM, Pashley CH, Slavin RG, Vijay HM, Wardlaw AJ. 2012. Fungi and allergic lower respiratory tract diseases. J Allergy Clin Immunol 129:280 –291. http://dx.doi.org/10.1016/j.jaci.2011.12.970. 6. Gilaberte M, Bartralot R, Torres JM, Reus FS, Rodríguez V, Alomar A, Pujol RM. 2005. Cutaneous alternariosis in transplant recipients: clinicopathologic review of 9 cases. J Am Acad Dermatol 52:653– 659. http://dx .doi.org/10.1016/j.jaad.2004.10.875. 7. Rid R, Onder K, MacDonald S, Lang R, Hawranek T, Ebner C, Hemmer W, Richter K, Simon-Nobbe B, Breitenbach M. 2009. Alternaria alternata TCTP, a novel cross-reactive ascomycete allergen. Mol Immunol 46:3476 –3487. http://dx.doi.org/10.1016/j.molimm.2009.07.024. 8. Bush RK, Prochnau JJ. 2004. Alternaria-induced asthma. J Allergy Clin Immunol 113:227–234. http://dx.doi.org/10.1016/j.jaci.2003.11.023. 9. Denning DW, Pashley C, Hartl D, Wardlaw A, Godet C, Del Giacco S, Delhaes L, Sergejeva S. 2014. Fungal allergy in asthma—state of the art and research needs. Clin Transl Allergy 4:14. http://dx.doi.org/10.1186 /2045-7022-4-14. 10. Mitakakis TZ, Barnes C, Tovey ER. 2001. Spore germination increases allergen release from Alternaria. J Allergy Clin Immunol 107:388 –390. http://dx.doi.org/10.1067/mai.2001.112602. 11. Gorbushina AA, Broughton WJ. 2009. Microbiology of the atmosphererock interface: how biological interactions and physical stresses modulate a sophisticated microbial ecosystem. Annu Rev Microbiol 63:431– 450. http://dx.doi.org/10.1146/annurev.micro.091208.073349. 12. Ragon M, Restoux G, Moreira D, Pape Møller A, López-García P. 2011. Sunlight-exposed biofilm microbial communities are naturally resistant to Chernobyl ionizing-radiation levels. PLoS One 6:e21764. http://dx.doi .org/10.1371/journal.pone.0021764. 13. Nosanchuk JD, Casadevall A. 2006. Impact of melanin on microbial virulence and clinical resistance to antimicrobial compounds. Antimicrob Agents Chemother 50:3519 –3528. http://dx.doi.org/10.1128/AAC.00545-06. 14. Jacobson ES. 2000. Pathogenic roles for fungal melanins. Clin Microbiol Rev 13:708 –717. http://dx.doi.org/10.1128/CMR.13.4.708-717.2000.

Antimicrobial Agents and Chemotherapy

aac.asm.org

1653

Fernandes et al.

15. Kimura N, Tsuge T. 1993. Gene cluster involved in melanin biosynthesis of the filamentous fungus Alternaria alternata. J Bacteriol 175:4427– 4435. 16. Thomma BP. 2003. Alternaria spp.: from general saprophyte to specific parasite. Mol Plant Pathol 4:225–236. http://dx.doi.org/10.1046/j.1364 -3703.2003.00173.x. 17. Kawamura C, Moriwaki J, Kimura N, Fujita Y, Fuji S, Hirano T, Koizumi S, Tsuge T. 1997. The melanin biosynthesis genes of Alternaria alternata can restore pathogenicity of the melanin-deficient mutants of Magnaporthe grisea. Mol Plant Microbe Interact 10:446 – 453. http://dx .doi.org/10.1094/MPMI.1997.10.4.446. 18. Kawamura C, Tsujimoto T, Tsuge T. 1999. Targeted disruption of a melanin biosynthesis gene affects conidial development and UV tolerance in the Japanese pear pathotype of Alternaria alternata. Mol Plant Microbe Interact 12:59 – 63. http://dx.doi.org/10.1094/MPMI.1999.12.1.59. 19. Kheder AA, Akagi Y, Akamatsu H, Yanaga K, Maekawa N, Otani H, Tsuge T, Kodama M. 2012. Functional analysis of the melanin biosynthesis genes ALM1 and BRM2-1 in the tomato pathotype of Alternaria alternata. J Gen Plant Pathol 78:30 –38. http://dx.doi.org/10.1007/s10327 -011-0356-4. 20. Cho Y, Davis JW, Kim KH, Wang J, Sun QH, Cramer RAJ, Lawrence CB. 2006. A high throughput targeted gene disruption method for Alternaria brassicicola functional genomics using linear minimal element (LME) constructs. Mol Plant Microbe Interact 19:7–15. http://dx.doi.org /10.1094/MPMI-19-0007. 21. Anitha R, Murugesan K. 2008. Melanin production in Alternaria helianthi. Arch Phytopathol Plant Prot 41:360 –364. http://dx.doi.org/10.1080 /03235400600796695. 22. Carzaniga R, Fiocco D, Bowyer P, O’Connell RJ. 2002. Localization of melanin in conidia of Alternaria alternata using phage display antibodies. Mol Plant Microbe Interact 15:216 –224. http://dx.doi.org/10.1094 /MPMI.2002.15.3.216. 23. Pihet M, Vandeputte P, Tronchin G, Renier G, Saulnier P, Georgeault S, Mallet R, Chabasse D, Symoens F, Bouchara JP. 2009. Melanin is an essential component for the integrity of the cell wall of Aspergillus fumigatus conidia. BMC Microbiol 9:177. http://dx.doi.org/10.1186/1471-2180 -9-177. 24. Tsai HF, Chang YC, Washburn RG, Wheeler MH, Kwon-Chung KJ. 1998. The developmentally regulated ALB1 gene of Aspergillus fumigatus: its role in modulation of conidial morphology and virulence. J Bacteriol 180:3031–3038. 25. Jahn B, Koch A, Schmidt A, Wanner G, Gehringer H, Bhakdi S, Brakhage AA. 1997. Isolation and characterization of a pigmentlessconidium mutant of Aspergillus fumigatus with altered conidial surface and reduced virulence. Infect Immun 65:5110 –5117. 26. Casadevall A, Rosas AL, Nosanchuk JD. 2000. Melanin and virulence in Cryptococcus neoformans. Curr Opin Microbiol 3:354 –358. http://dx.doi .org/10.1016/S1369-5274(00)00103-X. 27. Nosanchuk JD, Rosas AL, Lee SC, Casadevall A. 2000. Melanization of Cryptococcus neoformans in human brain tissue. Lancet 355:2049 –2050. http://dx.doi.org/10.1016/S0140-6736(00)02356-4. 28. Nosanchuk JD, Casadevall A. 2003. The contribution of melanin to microbial pathogenesis. Cell Microbiol 5:203–223. http://dx.doi.org/10 .1046/j.1462-5814.2003.00268.x. 29. Tanabe K, Park P, Tsuge T, Kohmoto K, Nishimura S. 1995. Characterization of the mutants of the Alternaria alternata Japanese pear pathotype deficient in melanin production and their pathogenicity. Ann Phytopathol Soc Jpn 61:27–33. http://dx.doi.org/10.3186/jjphytopath.61.27. 30. Hipólito E, Faria E, Alves AF, de Hoog GS, Anjos J, Gonçalves T, Morais PV, Estevão H. 2009. Alternaria infectoria brain abscess in a child with chronic granulomatous disease. Eur J Clin Microbiol Infect Dis 28: 377–380. http://dx.doi.org/10.1007/s10096-008-0623-2. 31. Anjos J, Fernandes C, Silva BM, Quintas C, Abrunheiro A, Gow NAR, Gonçalves T. 2012. ␤(1,3)-Glucan synthase complex from Alternaria infectoria, a rare dematiaceous human pathogen. Med Mycol 50:716 –725. http://dx.doi.org/10.3109/13693786.2012.675525. 32. Beltrán-García MJ, Prado FM, Oliveira MS, Ortiz-Mendoza D, Scalfo AC, Pessoa A, Jr, Medeiros MH, White JF, Di Mascio P. 2014. Singlet molecular oxygen generation by light-activated DHN-melanin of the fungal pathogen Mycosphaerella fijiensis in black Sigatoka disease of bananas. PLoS One 9:e91616. http://dx.doi.org/10.1371/journal.pone.0091616. 33. Kurtz MB, Heath IB, Marrinan J, Dreikorn S, Onishi J, Douglas C. 1994. Morphological effects of lipopeptides against Aspergillus fumigatus correlate with activity against (1,3)-␤-D-glucan synthase. Antimicrob

1654

aac.asm.org

34. 35.

36.

37. 38.

39. 40.

41.

42.

43.

44.

45. 46.

47. 48. 49. 50.

51.

Agents Chemother 38:1480 –1489. http://dx.doi.org/10.1128/AAC.38.7 .1480. Rajagopal K, Kathiravan G, Karthikeyan S. 2011. Extraction and characterization of melanin from Phomopsis: a phellophytic fungi isolated from Azadirachta indica A. Juss. Afr J Microb Res 5:762–766. Almeida-Paes R, Frases S, Sousa Araújo G, Evangelista de Oliveira MM, Gerfen GJ, Nosanchuk JD, Zancopé-Oliveira RM. 2012. Biosynthesis and functions of a melanoid pigment produced by species of the Sporothrix complex in the presence of L-tyrosine. Appl Environ Microbiol 78: 8623– 8630. http://dx.doi.org/10.1128/AEM.02414-12. Walker CA, Gómez BL, Mora-Montes HM, Mackenzie KS, Munro CA, Brown AJP, Gow NAR, Kibbler CC, Odds FC. 2010. Melanin externalization in Candida albicans depends on cell wall chitin structures. Eukaryot Cell 9:1329 –1342. http://dx.doi.org/10.1128/EC.00051-10. Wang Y, Aisen P, Casadevall A. 1996. Melanin, melanin “ghosts,” and melanin composition in Cryptococcus neoformans. Infect Immun 64: 2420 –2424. Dadachova E, Bryan RA, Huang X, Moadel T, Schweitzer AD, Aisen P, Nosanchuk JD, Casadevall A. 2007. Ionizing radiation changes the electronic properties of melanin and enhances the growth of melanized fungi. PLoS One 2:e457. http://dx.doi.org/10.1371/journal.pone.0000457. Pujol I, Aguilar C, Gené J, Guarro J. 2000. In vitro antifungal susceptibility of Alternaria spp. and Ulocladium spp. J Antimicrob Chemother 46:337–338. http://dx.doi.org/10.1093/jac/46.2.337. Fernandes C, Anjos J, Walker LA, Silva BM, Cortes L, Mota M, Munro CA, Gow NA, Gonçalves T. 2014. Modulation of Alternaria infectoria cell wall chitin and glucan synthesis by cell wall synthase inhibitors. Antimicrob Agents Chemother 58:2894 –2904. http://dx.doi.org/10.1128/AAC .02647-13. Wargenau A, Fleissner A, Bolten CJ, Rohde M, Kampen I, Kwade A. 2011. On the origin of the electrostatic surface potential of Aspergillus niger spores in acidic environments. Res Microbiol 162:1011–1017. http://dx .doi.org/10.1016/j.resmic.2011.07.006. Muszkieta L, Aimanianda V, Mellado E, Gribaldo S, Alcàzar-Fuoli L, Szewczyk E, Prevost MC, Latgé JP. 2014. Deciphering the role of the chitin synthase families 1 and 2 in the in vivo and in vitro growth of Aspergillus fumigatus by multiple gene targeting deletion. Cell Microbiol 16:1784 –1805. http://dx.doi.org/10.1111/cmi.12326. Chatterjee S, Prados-Rosales R, Frases S, Itin B, Casadevall A, Stark RE. 2012. Using solid-state NMR to monitor the molecular consequences of Cryptococcus neoformans melanization with different catecholamine precursors. Biochemistry 51:6080 – 6088. http://dx.doi.org/10.1021 /bi300325m. Chatterjee S, Prados-Rosales R, Tan S, Itin B, Casadevall A, Stark RE. 2014. Demonstration of a common indole-based aromatic core in natural and synthetic eumelanins by solid-state NMR. Org Biomol Chem 12: 6730 – 6736. http://dx.doi.org/10.1039/C4OB01066C. Nosanchuk JD, Casadevall A. 1997. Cellular charge of Cryptococcus neoformans: contributions from the capsular polysaccharide, melanin, and monoclonal antibody binding. Infect Immun 65:1836 –1841. Prados-Rosales R, Toriola S, Nakouzi A, Chatterjee S, Stark R, Gerfen G, Tumpowsky P, Dadachova E, Casadevall A. 2015. Structural characterization of melanin pigments from commercial preparations of the edible mushroom Auricularia auricula. J Agric Food Chem 63:7326 –7332. http://dx.doi.org/10.1021/acs.jafc.5b02713. Solano F. 2014. Melanins: skin pigments and much more—types, structural models, biological functions, and formation routes. New J Sci 2014: 498276. http://dx.doi.org/10.1155/2014/498276. Geis PA, Wheeler MH, Szaniszlo PJ. 1984. Pentaketide metabolites of melanin synthesis in the dematiaceous fungus Wangiella dermatitidis. Arch Microbiol 137:324 –328. http://dx.doi.org/10.1007/BF00410729. Gibello A, Ferrer E, Sanz J, Martin M. 1995. Polymer production by Klebsiella pneumoniae 4-hydroxyphenylacetic acid hydroxylase genes cloned in Escherichia coli. Appl Environ Microbiol 61:4167– 4171. Eisenman HC, Nosanchuk JD, Beau J, Webber WW, Emerson RJ, Camesano TA, Casadevall A. 2005. Microstructure of cell wall-associated melanin in the human pathogenic fungus Cryptococcus neoformans. Biochemistry 44:3683–3693. http://dx.doi.org/10.1021/bi047731m. van de Sande WW, de Kat J, Coppens J, Ahmed AO, Fahal A, Verbrugh H, van Belkum A. 2007. Melanin biosynthesis in Madurella mycetomatis and its effect on susceptibility to itraconazole and ketoconazole. Microbes Infect 9:1114 –1123. http://dx.doi.org/10.1016/j.micinf.2007.05.015.

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52. Kurahashi Y. 2001. Melanin biosynthesis inhibitors (MBIs) for control of rice blast. Pestic Outlook 12:32–35. http://dx.doi.org/10.1039/b100806o. 53. Diekema DJ, Messer SA, Hollis RJ, Jones RN, Pfaller MA. 2003. Activities of caspofungin, itraconazole, posaconazole, ravuconazole, voriconazole, and amphotericin B against 448 recent clinical isolates of filamentous fungi. J Clin Microbiol 41:3623–3626. http://dx.doi.org/10.1128/JCM.41 .8.3623-3626.2003. 54. Wetter TJ, Hazen KC, Cutler JE. 2005. Comparison between Aspergillus fumigatus conidia and hyphae susceptibilities to amphotericin B, itraconazole, and voriconazole by use of the mold rapid susceptibility assay. Med Mycol 43:525–532. http://dx.doi.org/10.1080/13693780500050796. 55. Valentine BP, Bainbridge BW. 1978. The relevance of a study of a temperature-sensitive ballooning mutant of Aspergillus nidulans defective in mannose metabolism to our understanding of mannose as a wall component and carbon/energy source. J Gen Microbiol 109:155–168. http://dx .doi.org/10.1099/00221287-109-1-155. 56. Bowman JC, Hicks PS, Kurtz MB, Rosen H, Schmatz DM, Liberator PA, Douglas CM. 2002. The antifungal echinocandin caspofungin acetate kills growing cells of Aspergillus fumigatus in vitro Antimicrob Agents Chemother 46:3001–3012. http://dx.doi.org/10.1128/AAC.46.9.3001-3012 .2002. 57. Perlin DS. 2007. Resistance to echinocandin-class antifungal drugs. Drug Resist Updat 10:121–130. http://dx.doi.org/10.1016/j.drup.2007.04.002. 58. Denning DW, Riniotis K, Dobrashian R, Sambatakou H. 2003. Chronic cavitary and fibrosing pulmonary and pleural aspergillosis: case series, proposed nomenclature change, and review. Clin Infect Dis 37(Suppl 3): S265–S280. http://dx.doi.org/10.1086/376526. 59. Chen SC, Slavin MA, Sorrell TC. 2011. Echinocandin antifungal drugs in

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60.

61.

62.

63.

64.

65.

fungal infections: a comparison. Drugs 71:11–14. http://dx.doi.org/10 .2165/11585270-000000000-00000. Fortwendel JR, Juvvadi PR, Perfect BZ, Rogg LE, Perfect JR, Steinbach WJ. 2010. Transcriptional regulation of chitin synthases by calcineurin controls paradoxical growth of Aspergillus fumigatus in response to caspofungin. Antimicrob Agents Chemother 54:1555–1563. http://dx.doi.org /10.1128/AAC.00854-09. Munro CA, Selvaggini S, de Bruijn I, Walker L, Lenardon MD, Gerssen B, Milne S, Brown AJ, Gow NAR. 2007. The PKC, HOG and Ca2⫹ signalling pathways co-ordinately regulate chitin synthesis in Candida albicans. Mol Microbiol 63:1399 –1413. http://dx.doi.org/10.1111/j.1365 -2958.2007.05588.x. Walker LA, Munro CA, de Bruijn I, Lenardon MD, McKinnon A, Gow NAR. 2008. Stimulation of chitin synthesis rescues Candida albicans from echinocandins. PLoS Pathog 4:e1000040. http://dx.doi.org/10.1371 /journal.ppat.1000040. Banks IR, Specht CA, Donlin MJ, Gerik KJ, Levitz SM, Lodge JK. 2005. A chitin synthase and its regulator protein are critical for chitosan production and growth of the fungal pathogen Cryptococcus neoformans. Eukaryot Cell 4:1902–1912. http://dx.doi.org/10.1128/EC.4.11.1902-1912 .2005. Wang Z, Zheng L, Hauser M, Becker JM, Szaniszlo PJ. 1999. WdChs4p, a homolog of chitin synthase 3 in Saccharomyces cerevisiae, alone cannot support growth of Wangiella (Exophiala) dermatitidis at the temperature of infection. Infect Immun 67:6619 – 6630. Nosanchuk JD, Ovalle R, Casadevall A. 2001. Glyphosate inhibits melanization of Cryptococcus neoformans and prolongs survival of mice after systemic infection. J Infect Dis 183:1093–1099. http://dx.doi.org/10 .1086/319272.

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