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Applied and Environmental Microbiology, January 2009, p. 493-503, Vol. 75, No. 2
0099-2240/09/$08.00+0     doi:10.1128/AEM.02077-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Production of Pyomelanin, a Second Type of Melanin, via the Tyrosine Degradation Pathway in Aspergillus fumigatus

Jeannette Schmaler-Ripcke,^1, Venelina Sugareva,^1,, Peter Gebhardt,² Robert Winkler,² Olaf Kniemeyer,¹ Thorsten Heinekamp,¹ and Axel A. Brakhage^1*

Department of Molecular and Applied Microbiology,¹ Department of Biomolecular Chemistry, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute and Friedrich Schiller University Jena, Beutenbergstrasse 11a, 07745 Jena, Germany²

Received 8 September 2008/ Accepted 14 November 2008

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Aspergillus fumigatus is the most important airborne fungal pathogen of immunosuppressed humans. A. fumigatus is able to produce dihydroxynaphthalene melanin, which is predominantly present in the conidia. Its biosynthesis is an important virulence determinant. Here, we show that A. fumigatus is able to produce an alternative melanin, i.e., pyomelanin, by a different pathway, starting from L-tyrosine. Proteome analysis indicated that the L-tyrosine degradation enzymes are synthesized when the fungus is grown with L-tyrosine in the medium. To investigate the pathway in detail, we deleted the genes encoding essential enzymes for pigment production, homogentisate dioxygenase (hmgA) and 4-hydroxyphenylpyruvate dioxygenase (hppD). Comparative Fourier transform infrared spectroscopy of synthetic pyomelanin and pigment extracted from A. fumigatus cultures confirmed the identity of the observed pigment as pyomelanin. In the hmgA deletion strain, HmgA activity was abolished and the accumulation of homogentisic acid provoked an increased pigment formation. In contrast, homogentisic acid and pyomelanin were not observed with an hppD deletion mutant. Germlings of the hppD deletion mutant showed an increased sensitivity to reactive oxygen intermediates. The transcription of both studied genes was induced by L-tyrosine. These results confirmed the function of the deleted genes and the predicted pathway in A. fumigatus. Homogentisic acid is the major intermediate, and the L-tyrosine degradation pathway leading to pyomelanin is similar to that in humans leading to alkaptomelanin.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Aspergillus fumigatus is the most important airborne human-pathogenic fungus. In immunocompromised patients, A. fumigatus has the potential to cause a life-threatening disease called invasive aspergillosis (IA). In the last two decades, the incidence of IA has increased tremendously. Specific diagnostics are still limited, as are therapeutic interventions. As a result, the mortality of IA is very high, ranging between 50 and 90% despite therapy (for an overview, see references 4, 38, 41, and 50).

A. fumigatus possesses specific physiological and molecular characteristics which make this fungus an aggressive pathogen (2, 41). These include the biosynthesis of a certain type of melanin, i.e., dihydroxynaphthalene (DHN) melanin, which is present in the gray-green conidia. Green fluorescent protein fusion with the polyketide synthase gene pksP, which is involved in DHN melanin biosynthesis, proved that this gene is transcribed in hyphae of germinating conidia isolated from lungs of infected immunosuppressed mice (31). PksP is also involved in the inhibition of phagosome-lysosome fusion and, thereby, the killing of A. fumigatus conidia (23). Additionally, DHN melanin has been shown to protect A. fumigatus from reactive oxygen intermediates (ROI) derived from host immune effector cells (21, 32). Therefore, this pigment biosynthesis pathway seems to contribute in a complex manner to the pathogenicity of A. fumigatus. Melanins are pigments of high molecular weight that are formed by oxidative polymerization of phenolic and/or indolic compounds with free radical-generating and -scavenging activity (42). They protect the fungus from oxidants (20), extreme temperatures (43), UV light (53), heavy metals (13, 14, 19), and antifungal agents (17). Previously, it was shown that melanins play an important role in the virulence of a broad range of pathogenic fungi (reviewed in references 19, 32, 37, and 49). Many fungi are able to synthesize black or brown pigments derived from L-tyrosine via dihydroxyphenylalanine (DOPA) (37, 55) in addition to DHN melanin. The DOPA-melanin pathway, in which tyrosinases or laccases hydroxylate tyrosine via DOPA to dopaquinone, which then auto-oxidizes and polymerizes, is the best-characterized melanization pathway from L-tyrosine (32). However, brown pigments may also be produced from L-tyrosine via a pathway involving the accumulation and auto-oxidation of intermediates of tyrosine catabolism (6, 7, 28). For instance, pyomelanins are synthesized from tyrosine through p-hydroxyphenylpyruvate (PHPP) and homogentisic acid (HGA) (7). The tyrosine degradation pathway has been subject to investigations in humans, as many severe metabolic disorders, e.g., phenylketonuria, alkaptonuria, tyrosinemia, and Hawkinsinuria, are associated with enzymatic defects in catabolism of phenylalanine and tyrosine (10, 35, 40). The genetic and biochemical basis of the inherited disorder alkaptonuria was elucidated largely by elegant experiments with the important model organism Aspergillus nidulans (11, 40). Pyomelanin and alkaptomelanin are merely different designations for the same pigment. However, the pigment produced by microbes is often referred to as pyomelanin, and the pigment produced by humans is called alkaptomelanin. The term pyomelanin was first introduced by Yabuuchi and Ohyama, who described a water-soluble brown pigment produced by the sanious bacterium Pseudomonas aeruginosa (56).

Although the synthesis of pyomelanin in a broad range of bacteria has been postulated, little is known about the tyrosine degradation pathway via HGA in clinically important fungi. Here, we showed for the first time that A. fumigatus is able to produce pyomelanin from L-tyrosine via HGA. We analyzed its biochemical and genetic basis.

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Strains and culture conditions.
A. fumigatus strains used in this study are listed in Table 1. Strain CEA17akuB^KU80 (8) was used as the wild type, unless otherwise noted, and to generate the hmgA and hppD deletion strains. A. fumigatus was cultivated in Aspergillus minimal medium (AMM) as described previously (54), containing 2% (wt/vol) agar for solid medium. Unless otherwise noted, 50 mM glucose was used as the carbon source. Liquid cultures were always grown at 37°C with shaking at 200 rpm. Pyrithiamine (Sigma-Aldrich, Germany) in a final concentration of 0.1 µg ml^–1 was added when selection for pyrithiamine resistance was required. For inhibition of HppD, sulcotrione (Riedel-de Haën, Germany) was used in a final concentration of 50 µmol liter^–1. For propagation of plasmids, Escherichia coli -Select chemically competent cells (Bioline, Germany) were employed and cultivated at 37°C in LB medium, supplemented with 100 µg ml^–1 ampicillin or 50 µg ml^–1 kanamycin.

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TABLE 1. Fungal strains used in this study

Standard DNA techniques.
Standard techniques for manipulation of DNA were carried out as described previously (45). Chromosomal DNA of A. fumigatus was prepared using a Master pure yeast DNA purification kit (Epicentre Biotechnologies). Southern blot analysis was carried out as previously described (16).

Two-dimensional gel electrophoresis.
A. fumigatus wild-type strain ATCC 46645 was preincubated for 14 h in AMM and further incubated for 55 h with and without the addition of 10 mM L-tyrosine. Proteome analysis of A. fumigatus mycelium extracts was essentially carried out according to the method described in reference 26. The absolute amount of 150 µg of protein was applied via anodic cup loading to rehydrated immobilized pH gradient strips with a nonlinear pH gradient from 3 to 11 (GE Healthcare Bio-Sciences, Germany). The second-dimension electrophoresis was performed with an Ettan DALTsix system (GE Healthcare Bio-Sciences, Germany). Preparative gels for mass analysis were stained with colloidal Coomassie blue (36), and for quantitative analysis, spots were made visible with a PlusOne silver staining kit (GE Healthcare Bio-Sciences). Images were analyzed with Image Master Platinum software (version 5.0). After background subtraction and normalization, spots were quantified using percent spot volumes, ratios, and gap parameters. Protein spots of interest were cut out and tryptically digested (47). Peptide mass and peptide fragment fingerprint spectra were measured by matrix-assisted laser desorption ionization-time of flight/time of flight mass spectrometry (Ultraflex 1; Bruker Daltonics, Germany) and subsequently identified by searching the fungi section of the NCBI database using the MASCOT interface (MASCOT 2.1.03; Matrix Science, United Kingdom) with the following parameters: Cys as an S-carbamidomethyl derivative and Met in oxidized form (variable), one missed cleavage site, and a peptide mass tolerance of 200 ppm. Hits were considered significant according to the MASCOT score (P < 0.05).

Generation of deletion mutants.
DNA fragments were amplified with Phusion high-fidelity DNA polymerase (Finnzymes, Finland) or Extender Polymerase Systeme (5 PRIME, Germany). To generate the hmgA knock-out plasmid, the hmgA gene, including 1.2-kbp upstream and downstream flanking regions, was amplified by PCR using oligonucleotides HmgAXbaI_for and HmgAXbaI_rev, introducing XbaI restriction sites (Table 2). The PCR product was cloned into plasmid pCR2.1 (Invitrogen, Germany), yielding pCR2.1hmgA. After digestion with XbaI, the 3,867-bp product was isolated and ligated into the single XbaI restriction site of plasmid pUC18 (Fermentas, Germany). The resulting plasmid, pUC18hmgA, was used as the template for an inverse PCR, employing the primers HmgASfiI_up and HmgASfiI_down, both containing an SfiI restriction site. Ligation of the SfiI-digested PCR fragment resulted in the generation of plasmid pUChmgA. The pyrithiamine resistance gene (ptrA) from plasmid pSK275 (gift from S. Krappmann), conferring pyrithiamine resistance (29), was inserted into the SfiI restriction site to yield pUC18hmgAptrA. The hmgAptrA sequence was amplified by PCR using oligonucleotides HmgAXbaI_for and HmgAXbaI_rev. The obtained PCR product was used for transformation of A. fumigatus. For deletion of the hppD gene, plasmid pCR2.1hppDptrA was generated. The hppD gene, including upstream and downstream flanking regions, was amplified from A. fumigatus genomic DNA using the oligonucleotides HppD_for and HppD_rev. The resulting 3,660-bp DNA fragment was cloned into plasmid pCR2.1. This plasmid was used as the template for the amplification with oligonucleotides HppD_Sfi_up and HppD_Sfi_down to modify the ends of the flanking regions with SfiI restriction sites and to remove the hppD coding sequence. After SfiI digestion of the PCR product, the ptrA gene was inserted as an SfiI fragment to give plasmid pCR2.1hppDptrA. PCR was performed employing pCR2.1hppDptrA as the template and oligonucleotides HppD_for and HppD_rev. The resulting DNA fragment, containing the ptrA gene flanked by hppD upstream and downstream regions, was used for transformation of A. fumigatus. Transformation of A. fumigatus was carried out using protoplasts as described previously (54).

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TABLE 2. Oligonucleotides used in this study

Complementation of hmgA and hppD mutants.
The hmgA and hppD mutant strains were complemented at the original gene locus by the use of modified wild-type genes. A dominant selection marker was not required, since hmgA and hppD mutant strains did not sporulate on AMM agar plates when L-tyrosine or L-phenylalanine was used as the sole carbon source. To distinguish between wild-type and complemented strains in Southern blot analysis, the nucleotide sequences of hppD and hmgA were slightly modified without affecting the protein sequence (see Results). To complement the hmgA mutant with the hmgA sequence, plasmid pUChmgA^C was generated. In this plasmid, an additional FspI restriction site was introduced into the hmgA gene by PCR by employing a FlipFlop site-directed mutagenesis kit (Bioline, Germany) with the help of oligonucleotides hmgA_FspI_for and hmgA_FspI_rev. For transformation of the A. fumigatus hmgA strain, a PCR fragment was generated, using pUChmgA^C as the template and oligonucleotides HmgAXbaI_for and HmgAXbaI_rev. To complement the deletion of the hppD strain, plasmid pCR2.1hppD^C was generated. In this plasmid, an additional XhoI restriction site was introduced into the hppD sequence by employing a FlipFlop site-directed mutagenesis kit and oligonucleotides HppD*_C_rev and HppD*_C_for the A. fumigatus hppD strain was transformed with a DNA fragment obtained by PCR with pCR2.1hppD^C as the template and oligonucleotides HppD_rev and HppD_for.

Susceptibility to H₂O₂ and diamide.
The sensitivities of the mutant strains to H₂O₂ and diamide compared with those of the wild-type strain were measured by agar plate diffusion assays. The 25-ml top and 25-ml bottom agar consisted of AMM supplemented with 50 mM glucose and 20 mM L-tyrosine. Conidia (2.5 x 10⁸) were added to the top agar and poured on top of the bottom agar in a petri dish. When the susceptibility of fresh conidia was tested, the oxidative insult was added immediately. To test the effect of H₂O₂ and diamide on germlings, agar plates were preincubated for 10 h at 37°C before the addition of the reagent. For both approaches, 150 µl of 5% (vol/vol) H₂O₂ or 200 µl of 0.2 M diamide was added via a hole with a diameter of 10 mm which was punched in the middle of the agar. Inhibition zones were measured 22 h after addition of the oxidative reagent. Eight replicates from three independent experiments served for the calculation of means and standard deviations.

Homogentisate dioxygenase assay.
HmgA activity was determined spectrophotometrically by measuring the formation of maleylacetoacetate at 330 nm as previously described (12). In brief, crude extracts were prepared from mycelia grown in liquid media. Conidia (1 x 10⁷) were inoculated in 100 ml AMM. After 20 h of preincubation, L-tyrosine was added, if necessary, to a final concentration of 10 mM. After a further 23 h of incubation, mycelia were harvested and either frozen in liquid nitrogen and stored or directly lysed by sonication in 50 mM potassium phosphate buffer, pH 7. After 15 min of centrifugation at 13,000 x g, the supernatant was used for enzyme assays containing 50 mM potassium phosphate buffer (pH 7), 2 mM ascorbate, 50 µM FeSO₄, 200 µM HGA, and crude protein extract at a concentration of 50 µg/ml. The substrate, HGA, was added just before measurement and after preincubation of the assay mixture for 10 min at 20°C. Homogentisate dioxygenase activity was calculated using the molar extinction coefficient of maleylacetoacetate, 13,500 M^–1 cm^–1 (46). Protein concentrations for calculation of specific activities were determined using a Coomassie Plus protein assay (Pierce Biotechnology).

FTIR analysis.
AMM (100 ml) with 50 mM glucose and 10 mM L-tyrosine was inoculated with 3 x 10⁸ A. fumigatus hmgA conidia. After cultivation for 53 h, the supernatant was filtered with Miracloth (Calbiochem) and precipitated overnight at room temperature by adjusting the pH to 2.0 with 1 M HCl. After centrifugation (16,100 x g, 20 min), the pellet was resuspended in 2.5 ml water at pH 12 and dialyzed in 3.5-kDa Slide-A-Lyzer dialysis cassettes (Pierce Biotechnology) against water, accompanied by a stepwise reduction of the pH from 10 to 7. The lyophilized pigment was then used for Fourier transform infrared spectroscopy (FTIR) analyses. In vitro-synthesized pyomelanin was used as the control in FTIR spectroscopy. Synthetic pyomelanin was produced by auto-oxidation of a 10 mM HGA solution at pH 10 with constant stirring for 3 days (adapted from reference 44). Polymerization was stopped, and precipitation was started by adjusting the pH to 2 with 6 M HCl. After precipitation overnight and centrifugation (16,100 x g, 20 min), the pellet was resuspended in 2.5 ml water at pH 12 and further treated as described above. The lyophilized sample was used for FTIR spectroscopy. Synthetic pyomelanin and melanin prepared from in vitro cultures were analyzed by using KBr disks with an FTIR spectrophotometer (FT/IR-4100; Jasco) equipped with a deuterated L-alanine triglycine sulfate detector.

Melanin formation and analysis of A. fumigatus cultures.
AMM (200 ml) was inoculated with 1 x 10⁷ conidia of each respective A. fumigatus strain. After 20 h of preincubation, L-tyrosine was added to a final concentration of 10 mM. Every 4 h, 4-ml samples were taken and filtered through Miracloth. Aliquots were stored at –20°C for further analyses. Pigment formation was analyzed by direct absorbance measurements of the supernatant of an alkalized (20 µl of 5 M NaOH per ml of sample) and centrifuged (16,000 x g for 2 min) sample at 405 nm. Another aliquot was acidified (20 µl of 10 M HCl per ml of sample), and the glucose concentration was determined by a chip sensor device (BIOSEN C_line, package GP+; EKF-diagnostic GmbH, Germany). Samples for HPLC were additionally filtered through Millex-LCR₁₃ filter units (0.5 µm; Millipore). Twenty microliters of the sample was injected into a Shimadzu high-pressure liquid chromatography (HPLC) system fitted with an RP₁₈ column (Eurospher 100C18, 250 mm by 4.6 mm, 5 µm; Merck, Germany). For elution, water with 0.1% (vol/vol) trifluoroacetic acid was used as buffer A and acetonitrile with 0.1% (vol/vol) trifluoroacetic acid was used as buffer B. Peaks were eluted at a flow rate of 1 ml min^–1 by applying the following gradient: 8% B for 12 min, gradient from 8% B to 95% B within 3 min, 95% B for 1 min, gradient from 95% B to 8% B within 2 min and, finally, 5 min at 8% B for recalibration. The compounds were detected with a Shimadzu LC-10 AD diode array detector at 280 nm. L-Tyrosine and HGA eluted under these conditions at 7.7 and 8.6 min, respectively. Concentrations of L-tyrosine and HGA were determined by calculations of peak areas at 280 nm and comparisons with standard curves for L-tyrosine and HGA, respectively. For instrument control, data acquisition, and data analysis, the CLASS-VP package (Shimadzu GmbH, Germany) was employed.

RNA isolation and first-strand cDNA synthesis.
For RNA isolation from in vitro-grown cultures, A. fumigatus was cultivated with and without 10 mM L-tyrosine as described for the homogentisate dioxygenase assay. Mycelium (100 mg) was used for RNA extraction employing a MasterPure yeast RNA purification kit (Epicentre Biotechnologies). After DNase treatment, 10 µg of total RNA was used for first-strand cDNA synthesis with SuperScript III reverse transcriptase and anchored oligo(dT)₂₀ primers (Invitrogen, Germany). Reverse transcription was conducted at 50°C for 3 h. RNA was hydrolyzed with 15 µl of 1 M NaOH for 10 min at 70°C and subsequently neutralized with 15 µl of 1 M HCl. After the addition of 6 µl of 3 M sodium acetate (pH 5.2), 2.5 µl of glycogen (5 mg/ml), and 200 µl of ice cold ethanol, cDNA was precipitated overnight at –20°C. After centrifugation for 30 min at 4°C and 16,000 x g, the pellet was washed with 70% (vol/vol) ethanol and resolubilized in 30 µl of Tris-HCl buffer, pH 8.0.

Reverse transcription-PCR analysis.
cDNA synthesis was used to determine transcription levels of hmgA and hppD compared with those of the constitutively formed citrate synthase (Afu5g04230, citA) transcripts according to the method described in reference 18. For this purpose, oligonucleotides AfCitAcode_up and AfCitAcode_down were used for the amplification of a 575-bp fragment of citA transcripts. Oligonucleotides AfHppDcode2_for and AfHppDcode2_rev allowed the specific amplification of a 591-bp region of hppD transcript. With oligonucleotides AfHmgAcode_up and AfHmgAcode_down, a 563-bp DNA fragment of the coding region of hmgA could be detected. Transcript amplification using GoTaq DNA polymerase (Promega, Germany) was carried out with a Veriti fast thermal cycler (Applied Biosystems) with 5 ng cDNA isolated from in vitro-grown cultures as the template. As the control, 0.1 ng genomic DNA was applied under the same conditions.

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Addition of L-tyrosine leads to the production of a pyomelanin in A. fumigatus catalyzed by the tyrosine degradation pathway.
A. fumigatus produced a brown pigment when grown in AMM supplemented with L-tyrosine. This brown compound accumulated in the hyphae but was also excreted into the medium (Fig. 1A). Since A. fumigatus is known to produce DHN melanin (22, 30, 51), we tested whether the biosynthesis of the pigment is connected to that of DHN melanin. Therefore, two A. fumigatus mutant strains deficient in DHN melanin biosynthesis were analyzed. The A. fumigatus pksP mutant strain produces white conidia due to a mutation in the PksP polyketide synthase that is essential for DHN melanin biosynthesis (30). The abr2 strain also has a defect in DHN melanin biosynthesis due to deletion of a laccase (48). Interestingly, both DHN melanin mutant strains, the pksP mutant and the abr2 strain, were able to produce the brown pigment induced by the addition of L-tyrosine to the medium (see Fig. 3D for the pksP mutant phenotype), excluding the possibility that the brown pigment represented some DHN melanin derivative.

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FIG. 1. Pigment formation and proteome analysis of A. fumigatus grown with (+Tyr) and without (–Tyr) L-tyrosine. The wild-type strain was preincubated for 15 h in AMM and then further incubated for 55 h with and without the addition of 10 mM L-tyrosine. (A) Pigment formation was observed for A. fumigatus mycelia and in the medium. (B) Two-dimensional gel electrophoresis of proteins extracted from A. fumigatus hyphae. Silver-stained protein pattern of cytoplasmic proteins from A. fumigatus grown with and without the addition of 10 mM L-tyrosine. Selected protein spots with increased levels are highlighted by numbered arrows. They correspond to the proteins listed in Table 3.

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FIG. 3. (A and B) Deletion and complementation of hppD and hmgA. (A) Schematic representation of the chromosomal hppD locus in the wild type (WT), the hppD deletion mutant (), and the complemented (C) strain. Southern blot analysis proves the deletion of the hppD gene and the reintroduction of the point mutation-carrying hppD gene containing an additional XhoI restriction site. M denotes the lane with the Gene Ruler 1-kb DNA ladder (Fermentas, Germany). (B) Schematic representation of the chromosomal hmgA locus in the WT, the hmgA deletion mutant (), and the complemented (C) strain. Southern blot analysis verified the deletion of the hmgA gene and the reintroduction of the point mutation-carrying hmgA gene with an extra FspI site. Lane M depicts HyperLadder I (Bioline GmbH, Germany). Restriction endonuclease cleavage sites, the DNA fragments identified by Southern blot analysis, and the positions to which the probe hybridizes are indicated. (C) Phenotypes of the wild-type (WT), hmgA, hppD, and complemented mutant strains grown for 68 h on AMM agar plates with 50 mM glucose (left panel) and without glucose but with 10 mM L-tyrosine as the sole carbon source. The growth on AMM agar plates with 50 mM glucose and 10 mM PHPP or, alternatively, 10 mM HGA is also shown. (D) Pigment production of wild-type, hmgA, hppD, complemented, and pksP mutant strains grown in AMM with L-tyrosine. The strains were cultivated for 64 h in 50 ml of AMM supplemented with 10 mM L-tyrosine and with an inoculum of 5 x 107 conidia. The upper panel displays the culture permeates and the lower panel the mycelia. (E) Quantification of pigment production by absorption measurements at 405 nm. CEA17akuBKU80 wild-type and hmgA mutant strains were inoculated with 1 x 105 conidia/ml for 68 h in AMM (white bar), AMM with 10 mM L-tyrosine (gray bar), AMM with 10 mM L-phenylalanine (blue bar), and AMM with 10 mM phenylacetate (black bar). The experiment was repeated three times in duplicate. Standard deviations are calculated from two independent experiments.

Proteome analysis applying two-dimensional gel electrophoresis of mycelia grown in the presence of tyrosine (Fig. 1B) led to the identification of proteins that were assigned to tyrosine catabolism (Table 3, shown in bold). These proteins were candidates likely to be involved in the degradation of L-tyrosine to HGA that can polymerize to pyomelanin (Fig. 2A). By a BLAST search of the TIGR database (http://www.tigr.org/tdb/e2k1/afu1/), the genes putatively involved in tyrosine degradation were deduced from the identified proteins. Aspartate transaminase catalyzes the deamination of L-tyrosine to p-hydroxyphenylalanine. The tyrosine degradation product fumarate can be introduced into the citrate circle, where it is metabolized to oxaloacetate via malate dehydrogenase after conversion to malate by fumarate hydratase, which explains the upregulation of malate dehydrogenase. The other two identified genes form a cluster with four further genes (Fig. 2B). Database annotations predicted three introns for both the hppD and hmgA genes. It is interesting that the AFUA_2g04210, fahA, and maiA genes apparently do not contain any introns. The AFUA_2g04210 gene encodes a conserved hypothetical protein of unknown function. A putative transcription factor containing a fungal Zn₂Cys₆ binuclear cluster domain (AFUA_2g04262) is located 1,071 bp upstream of the maiA gene. The gene cluster shows similarity to the respective cluster in A. nidulans, which was shown to be involved in tyrosine degradation (11). However, in A. nidulans, there is an additional putative gene (AN 1894.3) of unknown function between the gene encoding a putative transcription factor (AFUA_2g04262) and the maleylacetoacetate isomerase gene, which is absent in the A. fumigatus genome. The other upregulated proteins, NADH-quinone oxidoreductase, glyceraldehyde 3-phosphate dehydrogenase, aldehyde dehydrogenase, and alcohol dehydrogenase, may also be involved in tyrosine metabolism. Nucleoside diphosphate kinase and peptidyl-prolyl cis-trans isomerase are rather unspecifically upregulated due to secondary effects.

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TABLE 3. Proteins with increased levels in A. fumigatus cultures grown in AMM after supplementation with 10 mM L-tyrosine

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FIG. 2. (A) L-Tyrosine degradation pathway (modified from reference 35). The enzymes involved are indicated in bold as follows: PhhA, phenylalanine hydroxylase; Tat, tyrosine aminotransferase; HppD, 4-hydroxyphenylpyruvate dioxygenase; HmgA, homogentisate dioxygenase; MaiA, maleylacetoacetate isomerase; FahA, fumarylacetoacetate hydrolase. Homogentisate can lead to the production of pyomelanin through benzoquinone acetic acid after oxidation (O) and polymerization. (B) Organization of the L-tyrosine degradation gene cluster in A. fumigatus. Genes are illustrated as arrows. Locus tags, transcript sizes, numbers of introns, putative protein function, and the number of amino acids of the deduced proteins are listed.

Deletion of hppD and hmgA impairs growth on L-tyrosine as the sole carbon source and affects pyomelanin formation.
To verify the predicted pathway and to analyze the effect of accumulation of HGA on the pigment formation of A. fumigatus, the two central genes of the pathway, hmgA and hppD, were deleted. HppD catalyzes the formation of HGA, whereas homogentisate dioxygenase is involved in the cleavage of the aromatic ring of HGA and its conversion to maleylacetoacetate (Fig. 2A). For deletion of hmgA and hppD in A. fumigatus, the coding sequence of each gene was replaced by ptrA. For complementation of the deletion mutants, a strategy based on homologous recombination was applied using the wild-type gene in which a point mutation was inserted. In the hmgA gene, thymine at position 1248 was replaced by guanine. This base exchange did not alter the amino acid sequence but introduced an FspI restriction site that enabled differentiation between wild-type and complemented mutant strains by Southern blot analysis. For complementation of the hppD gene, cytosine at position 1022 was replaced by thymine, introducing an XhoI restriction site. Selection of complemented strains was achieved by using AMM agar plates containing phenylalanine as the sole carbon source. After transformation, complemented strains were able to produce conidia, in contrast to the deletion strains with phenylalanine as the sole carbon source, which did not sporulate (data not shown; see Fig. 3C).

The integration of the ptrA gene in the locus of the hppD and hmgA genes and the reintroduction of wild-type genes are shown in Fig. 3A and B. In the hmgA mutant strain, the band of 4.5 kbp, characteristic of the wild type, had disappeared. Instead, an 8.2-kbp DNA fragment was visible, indicative of the ptrA-encoding DNA fragment and thus the replacement of the wild-type gene with the ptrA gene. In the hmgA complemented strain, the expected band of 2.1 kbp was visible. The hmgA deletion strain and the complemented strain were designated the hmgA and hmgA^C strains, respectively. In the hppD deletion strain, the band at 2.2 kbp, which is characteristic of the wild-type strain, had disappeared. Instead, the fragment at 3.6 kbp appeared. In the complemented strain, only the characteristic fragment at 1.5 kbp was detectable. These strains were referred to as the hppD and hppD^C strains in further studies. The phenotypes of the hmgA and hppD strains were characterized with different media (Fig. 3C). On AMM agar plates, no differences in growth of the deletion strains and wild-type strains were apparent (Fig. 3C, left panel). The addition of 10 mM L-tyrosine to AMM liquid cultures (Fig. 3D) stained the mycelia and culture media of wild-type, complemented, and pksP mutant strains light brown, whereas in the hmgA mutant, the color was dark brown. In contrast, no pigment production was observed for the hppD strain. An examination of pigment release in liquid cultures (Fig. 3E) indicated that besides tyrosine, phenylalanine and phenylacetate also provoke pigment formation. Tyrosine addition led to the highest pigment formation in wild-type cultures, which is the reason tyrosine was used in most experiments as the inducer. Furthermore, when tyrosine was given as the sole carbon source, the growth rate of the hppD strain was reduced on agar plates, the mycelia of the hppD and hmgA strains were less dense, conidiation was severely impaired, and tyrosine crystals in the media were not consumed, in contrast to observations for wild-type and complemented strains (Fig. 3C). Still, the pigment was clearly visible in the hmgA deletion strain due to residual mycelial growth based on carbon sources present in the agar. The same was true for the growth on L-phenylalanine as the sole carbon source (data not shown). The severe growth defects on AMM agar plates with tyrosine or phenylalanine as the sole carbon source confirmed the involvement of HmgA and HppD in tyrosine degradation.

Additionally, the strains were incubated on AMM agar plates supplemented with HGA or PHPP. The addition of HGA, the accumulation product of the hmgA strain, did not impair growth; therefore, pyomelanin formation is not a result of detoxification of HGA. However the addition of PHPP, the accumulation product of the hppD mutant, led to reduced growth and sporulation of the wild-type strain, even more so with the deletion strains. Consequently, PHPP needs to be degraded for detoxification. Integration of a functional gene cured the defects of the deletion strains on all applied media, demonstrating that the phenotypes are a direct result of the loss of HmgA and HppD activity.

Pigment results from polymerization of HGA.
FTIR spectroscopy was used to analyze the pigment, which accumulated in cultures of the hmgA strain, since it is regarded as the most informative method for structural analysis of melanins (3). The overlay of the FTIR spectra of synthetic pyomelanin and pigment extracted from fungal cultures showed a high degree of similarity (Fig. 4). Both spectra depict a broad absorption at 3,420 cm^–1, which is due to associated or polymeric OH groups. The stretching vibrations for aliphatic CH bonding appear at 2,952 cm^–1 and 2,925 cm^–1 for natural and synthetic melanin, respectively. At 1,586 cm^–1, the symmetric carboxylate stretching vibrations (COO^–) are detectable according to the basic isolation procedure. The fingerprint regions between 1,450 cm^–1 and 650 cm^–1 resemble each other closely. The high level of identity between the synthetic pyomelanin and the pigment extracted from fungal cultures indicates that the hmgA mutant produces pyomelanin when cultivated with L-tyrosine.

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FIG. 4. FTIR spectrum overlay from synthetic HGA melanin and lyophilized extracellular pigment extracted from hmgA culture.

HGA accumulation correlates with L-tyrosine consumption and pyomelanin formation.
To provide evidence for pigment formation from L-tyrosine via the accumulation of HGA, tyrosine catabolism in A. fumigatus liquid cultures was analyzed. Melanin accumulation was observed by measuring absorption at 405 nm. L-Tyrosine and HGA concentrations were determined by HPLC analysis. In cultures grown without L-tyrosine, pigment accumulation and HGA formation were lacking (Fig. 5A). For wild-type and hmgA AMM tyrosine cultures, the consumption of L-tyrosine and the formation of HGA were clearly visible (Fig. 5B and E). In the hmgA strain, HGA concentration as well as absorption values were even higher than those of the wild type. In this mutant, HGA accumulated because HGA could not be cleaved to maleylacetoacetate (Fig. 2A). hmgA cultures showed a decelerated decrease in L-tyrosine concentration, probably due to a feedback inhibition via accumulation of HGA. The analysis of wild-type cultures grown with the HppD inhibitor sulcotrione (9) led to results very similar to those for the hppD mutant, i.e., HGA formation was not detected and absorption hardly increased (Fig. 5C and D). Sulcotrione (50 µM) did not inhibit growth of the wild-type strain but prevented formation of pigment when cultivated with 10 mM L-tyrosine.

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FIG. 5. Pigment formation, HGA synthesis, and L-tyrosine consumption in different A. fumigatus strains. The wild type (WT) with (C) and without (B) the Hppd inhibitor sulcotrione and hppD (D) and hmgA (E) strains were precultivated with 1 x 107 conidia in 200 ml AMM for 20 h prior to the addition of L-tyrosine. Additionally, the wild type was cultivated without tyrosine for comparison (A). Pigment formation is reflected by an increase in absorption at 405 nm. HGA and L-tyrosine concentrations were measured by HPLC. (F) Glucose consumption and pH changes of the cultures mentioned above were monitored.

Additionally, glucose consumption and changes in pH values were monitored. Glucose was completely exhausted after 40 to 50 h of cultivation. An increase in pH was detectable at the same time (Fig. 5F). Therefore, the strains showed very similar growth rates in liquid cultures. This is an important prerequisite for the analysis of pigment formation, since high pH values accelerate the process and glucose apparently can repress tyrosine catabolism in Yarrowia lipolytica (6).

The complemented hmgA^C and hppD^C strains gave the same data as the wild-type strain (data not shown). Taken together, these experiments proved the inability of the hmgA strain to degrade HGA and of the hppD strain to form HGA. Thus, it was verified that hppD and hmgA code for 4-hydroxyphenylpyruvate dioxygenase and homogentisate dioxygenase, respectively. Moreover, the data show a positive correlation between HGA accumulation and melanin formation.

Tyrosine catabolism is involved in the oxidative-stress response of A. fumigatus.
To assess the role of metabolites of the tyrosine degradation in the resistance of A. fumigatus to ROI, an agar plate diffusion assay was employed. The sensitivities of hmgA and hppD mutant strains to H₂O₂ and the thiol-oxidizing agent diamide were compared with those of the wild-type and complemented strains (Fig. 6A and B). The direct confrontation of freshly harvested conidia with both oxidative agents revealed no differences between the strains. To test the sensitivity of germlings, conidia were preincubated for 10 h in AMM before the addition of H₂O₂ and diamide, respectively. Under these conditions, the hppD strain showed a significant (P < 0.05) increase in ROI sensitivity compared with that of the other strains tested. Pigmentation of hyphae and media was absent only in the hppD strain. This finding implies a role of pyomelanin as a protective agent against peroxides and thiol-oxidizing agents. In all strains except for the hppD deletion mutant, mycelia were dark colored and melanin was released into the medium (Fig. 6A, lower panel). The pigment accumulated particularly in the border of the inhibition zones, most evidently in the hmgA strain assay, which implied a supporting role for H₂O₂ in pigment formation.

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FIG. 6. Sensitivity of hppD and hmgA mutant strains to H2O2 (A) and diamide (B) in an inhibition zone assay. hppD and hmgA deletion strains were compared with the wild-type (WT) and complemented strains. Black bars indicate the inhibition zones in the assay with fresh conidia, gray bars the inhibition zones with germinated conidia. In all experiments, AMM supplemented with 20 mM L-tyrosine was used. Error bars indicate the standard deviations for three independent experiments with eight replicates. * denotes significantly different (P < 0.05) from the wild type. (A) Lower panel, induction of pigmentation on the bottom side in the experiment with preincubation. (C) HmgA activity in hppD and hmgA mutant strains compared with wild-type and complemented strains. Enzyme-specific activity of mycelia grown with or without 10 mM tyrosine in the medium was determined.

Despite the strong pigmentation of the hmgA mutant strain, inhibition of growth was comparable to that of the wild-type strain. Consequently, the increase of pigmentation due to the accumulation of HGA in the hmgA strain did not further increase resistance to ROI. To confirm that the above-mentioned effects derived from the deletion of the hmgA and hppD genes, the complemented mutant strains were studied. Both complemented mutant strains revealed inhibition zones similar to that of the wild-type strain in both assays.

Transcription of hmgA and hppD is induced by tyrosine.
A semiquantitative transcript analysis by reverse transcription-PCR allowed the comparison of hppD und hmgA mRNA steady-state levels of mycelia grown with and without tyrosine. In cultures grown in AMM without L-tyrosine, mRNA steady-state levels of the genes hppD and hmgA were low in comparison to those of the citA reference gene (Fig. 7). In contrast, the steady-state mRNA level of hmgA and hppD was strongly increased by the addition of L-tyrosine. This finding correlated well with the increase of HmgA activity upon the addition of L-tyrosine to the medium (Fig. 6C) and shows that the genes are induced by tyrosine at the transcriptional level.

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FIG. 7. Semiquantitative analysis of hmgA and hppD transcripts. (A) Analysis of the wild-type (WT) strain grown in AMM with (+Tyr) and without (–Tyr) the addition of 10 mM L-tyrosine. Lane M, 100-bp DNA ladder; gDNA, genomic DNA; cDNA, first-strand synthesized cDNA; citA, transcript of citrate synthase used as the control; hppD, transcript of PHPP dioxygenase; hmgA, transcript of HGA dioxygenase. citA showed a constant mRNA steady-state level and served as the reference for expression levels. (B) Scheme of the expected fragment sizes for citA, hppD, and hmgA. White bars indicate introns. Since the amplified regions from genomic DNA include introns, the PCR products for cDNA are smaller.

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One of the characteristics of A. fumigatus is the production of gray-green conidia. The pigment consists of a DHN melanin pigment (reviewed in reference 32). Here, we showed for the first time that A. fumigatus is able to produce another type of pigment, which was identified by genetic, biochemical, and chemical means as pyomelanin. This compound derived from L-tyrosine via a tyrosine degradation pathway. Further arguments for the presence of this pathway derive from the converse phenotype of the hppD mutant compared to a pksP mutant deficient in the polyketide synthase essential for DHN melanin formation. The pksP mutant strain produced white conidia and released a brown pigment when cultivated with L-tyrosine. In contrast, the hppD strain produced pigmented conidia, but no pigment accumulation occurred in liquid cultures supplemented with L-tyrosine. Moreover, the addition of the HppD inhibitor sulcotrione to the medium completely abolished pyomelanin formation by A. fumigatus, further confirming that the tyrosine degradation pathway is responsible for pyomelanin formation. The detection of HGA in culture supernatants led to the conclusion that the pigment is synthesized extracellularly and that the darkened mycelia show the deposition of the pigment on the surface of hyphae. Based on these results, A. fumigatus has the ability to produce two different melanins, i.e., DHN melanin pigment via the polyketide biosynthesis pathway (30, 51) and pyomelanin via L-tyrosine degradation. DHN melanin was found in many different fungi, both pathogens and nonpathogens (reviewed in references 32 and 55). Until now, pyomelanin has rarely been detected in filamentous fungi. In A. nidulans, the pathway was studied to elucidate the molecular basis of inherited diseases of humans involving the formation of black pigments, such as alkaptonuria. Studies of A. nidulans and the transfer of results to humans showed that alkaptonuria is associated with enzymatic defects in the catabolism of phenylalanine and tyrosine (10, 40). Studies of an A. nidulans hmgA deletion mutant (11) bear some analogy to our results with A. fumigatus. We focused on the degradation of tyrosine since it is a closer precursor of HGA than phenylalanine and induced stronger pigmentation in the wild-type strain, although the two compounds seem to cause similar effects. As shown for A. nidulans, A. fumigatus contains only a single functional gene coding for homogentisate dioxygenase. The same is true for PHPP dioxygenase. The pigmentation is induced by tyrosine, phenylalanine, and phenylacetic acid, which agrees well with observations for A. nidulans. It will be interesting to compare the regulation of the tyrosine degradation pathway in the two organisms in order to find out whether a basic principle in the difference of virulence of the two strains relies on pyomelanin production. Also, the coexistence of DHN melanin and pyomelanin in A. fumigatus (A. nidulans does not produce DHN melanin) might be an interesting feature with regard to virulence.

In the thermodimorphic fungus Paracoccidioides brasiliensis, which causes paracoccidioidomycosis, genes involved in tyrosine degradation were identified during a microarray analysis. The hppD gene was highly expressed during yeast mycelium transition. Addition of an HppD inhibitor inhibited growth and differentiation of the pathogenic yeast phase of the fungus in vitro (39).

The genes hppD, hmgA, AFUA_2g04210, fahA, and maiA, which are involved in tyrosine degradation in A. fumigatus, form a cluster located on chromosome 2. A similar cluster is found in A. nidulans (comparative data analysis at http://www.tigr.org/tigr-scripts/sybil-asp/sybilHome.pl?db=asp). Clustering of genes encoding proteins involved in the same catabolic pathway is not uncommon for filamentous fungi. Enzymatic and regulatory genes for other well-studied catabolic pathways, such as quinate, ethanol, proline, and nitrate utilization, were found to be clustered in various filamentous fungi (reviewed in reference 25). Furthermore, fungal genes involved in secondary metabolism biosynthesis are clustered (5). These clusters also include genes involved in DHN melanin biosynthesis in A. fumigatus (30, 52). The existence of the tyrosine degradation cluster in A. fumigatus suggests that the genes of this cluster are regulated in a common manner. This is supported by the finding that both genes studied here in more detail, hmgA and hppD, were shown to be induced at the transcriptional level by L-tyrosine. It is unclear yet how this tyrosine regulation is mediated. Possible regulatory genes, AFUA_2G04210 and AFUA_2G04262, are part of the cluster. It has been shown previously that bacterial homogentisate clusters possess at least one regulatory protein. For example, in Pseudomonas putida, the hmgR regulatory gene located upstream of hmgA, fahA, and maiA codes for an IclR-type regulator, which acts as a repressor of an aromatic catabolic pathway. It controls the inducible expression of the genes in the homogentisate cluster. HGA is the inducer molecule (1). Further studies will show how the tyrosine signal is transmitted in A. fumigatus.

Here, we showed that in A. fumigatus, tyrosine degradation is dispensable during growth on minimal agar plates with glucose as the carbon source but essential for growth on agar plates containing L-tyrosine as the sole carbon source. The visible production of pyomelanin requires L-tyrosine or L-phenylalanine in the medium.

It seems likely that L-tyrosine is also present in the lungs of immunosuppressed patients, allowing A. fumigatus to produce pyomelanin during infection. Consistently, A. fumigatus secretes a variety of proteases (34) during colonization of the lung tissue. It was proposed that protein degradation of the lung tissue is essential for host invasion (27). Indeed, this was proven by the finding that mutants in the methylcitrate synthase of A. fumigatus exhibited strongly attenuated virulence due to the accumulation of toxic propionyl-coenzyme A, which derives from the degradation of isoleucine, valine, or methionine. Thus, amino acids are available and serve as nutrient sources during invasive growth (18). This argument is also supported by the finding that L-tyrosine is present in sera at a concentration of between 21 and 107 µM (15) to serve as precursor for a variety of biocompounds, e.g., catecholamines, thyroid hormones, and DOPA melanin. It is also available in tissue due to protein degradation. Furthermore, it can be synthesized from L-phenylalanine by the phenylalanine hydroxylase.

A current model concerning pathogenicity of A. fumigatus involving melanin is that conidia are protected from ROI to a certain extent by the DHN melanin pigment and that its biosynthesis genes are also involved in the production of an immunosuppressive compound (4). When conidia germinate and hyphae grow out, e.g., from macrophages, it is conceivable that the formation of pyomelanin further protects the germlings and outgrowing hyphae from ROI, e.g., produced by neutrophils. Consistently, as shown here, by comparing mutant strains with the wild type during growth on agar plates supplemented with L-tyrosine, we found that the hppD strain, which lacks pyomelanin, displayed a higher susceptibility to ROI. Similar findings were previously reported for Burkholderia cenopacia, and pyomelanin was shown to protect the bacterium from in vitro and in vivo sources of oxidative stress (24). However, the role of ROI in the killing of A. fumigatus has been recently challenged since the deletion mutant of the main regulator for the ROI response of A. fumigatus, AfYap1, did not influence the killing of A. fumigatus by immune effector cells (33). Additionally, as shown here, the hmgA deletion mutant characterized by enhanced pyomelanin formation did not show reduced sensitivity to ROI. Therefore, the role of pyomelanin in the scavenging of ROI in vivo remains to be elucidated. Further experiments will show whether the ability to produce pyomelanin influences the pathogenic potential of A. fumigatus.

 
Kim Langfelder and Burghard Liebmann are gratefully acknowledged for initial experiments. We thank Nancy Hannwacker, Silke Steinbach, Ingrid Richter, and Sophia Keller for their excellent technical assistance. We are particularly indebted to Olaf Scheibner for the identification of proteins by mass spectrometry.

This research was supported by the Deutsche Forschungsgemeinschaft (SPP 1160) and the Hans-Knöll-Institute.

 
* Corresponding author. Mailing address: Department of Molecular and Applied Microbiology, Leibniz Institute for Natural Product Research and Infection Biology, Hans Knöll Institute and Friedrich Schiller University Jena, Beutenbergstrasse 11a, 07745 Jena, Germany. Phone: 49 (0)3641-532 1001. Fax: 49 (0)3641-532 0802. E-mail: axel.brakhage{at}hki-jena.de

Published ahead of print on 21 November 2008.

J.S.-R. and V.S. contributed equally to this work.

Present address: Department of Medical Microbiology and Hospital Hygiene, University Hospital Rostock, Schillingallee 70, 18057 Rostock, Germany.

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Applied and Environmental Microbiology, January 2009, p. 493-503, Vol. 75, No. 2
0099-2240/09/$08.00+0     doi:10.1128/AEM.02077-08
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