Michael H. Wheeler

Melanins and Their Importance in Pathogenic Fungi

Melanins are generally described as dark brown or black pigments of high molecular weight formed by oxidative polymerization of phenolic compounds. Certain yellow, red, green, purple, or blue pigments have similar chemical structures and occasionally are referred to as types of melanins. Melanins are found in humans and various other warm- and cold-blooded vertebrates; invertebrates, including insects; higher plants; fungi; and bacteria, including actinomycetes. Most animal melanins are synthesized by tyrosinase, whereas a number of less specific polyphenol oxidases may form melanins in various cellular and extracellular environments of other organisms. In some cases melanins are autoxidative products made in the absence of enzymes.

Comparisons of fungal melanin biosynthesis in ascomycetous imperfect and basidiomycetous fungi

Melanin biosynthesis was studied in 26 species of fungi belonging to 16 genera using the melanin inhibitor tricyclazole (5-methyl-1,2,4-triazolo(3,4 b )-benzothiazole) and enzyme reactions in cell-free homogenates. All but one of the 20 dark brown and black ascomycetous and imperfect fungi apparently made melanin from 1,8-dihydroxynaphthalene. Cell-free homogenates of these fungi metabolized two or more melanin substrates (1,3,6,8-tetrahydroxynaphthalene, (+)-scytalone, 1,3,8-trihydroxynaphthalene or vermelone) to intermediates in the melanin pathway, including 1,8-dihydroxynaphthalene, and/or the melanin biosynthetic pathway in these fungi was blocked in vivo by tricyclazole, causing a change in fungal colour and an accumulation of 2-hydroxyjuglone, flaviolin, and other melanin shunt products. Aspergillus niger and six basidiomycetes did not metabolize these melanin substrates, and melanin biosynthesis in these fungi was not affected by tricyclazole. Thus, many ascomycetous and related imperfect fungi appear to make melanin from 1,8-dihydroxynaphthalene, whereas basidiomycetes and at least some imperfect fungi probably use alternative pathways.

Pentaketide metabolites of melanin synthesis in the dematiaceous fungus Wangiella dermatitidis

Melanin synthesis in the dematiaceous, polymorphic hyphomycete Wangiella dermatitidis, a human pathogen, was investigated by biochemical and physiological techniques. Mutants with a decrease or loss in melanin synthesis were induced and isolated. Melanin precursors were obtained from the mutants, purified, and then identified by comparison with authentic compounds from Verticillium dahliae. Isolation of scytalone, vermelone, flaviolin, and 1,8-dihydroxynaphthalene from the mutants of Wangiella dermatitidis, and cross-feeding of the mutants with those of Verticillium dahliae indicated that melanin synthesis in this organism took place by the pentaketide pathway. Melanin that formed in cell walls of an albino mutant treated with scytalone was identical in appearance to that in cell walls of the wild-type strain. This also suggested that pentaketide synthesis of melanin occurred in the fungus.

Melanin biosynthesis in Thielaviopsis basicola

An albino mutant of Thielaviopsis basicola metabolized scytalone, vermelone, and 1,8-dihydroxynaphthalene to melanin like that in the wild-type fungus. The melanin appeared as electron-dense granular material localized in the cell walls of hyphae and chlamydospores. The albino mutant also converted catechol and l -3,4-dihydroxyphenylalanine to dark pigments, but these were different from wild-type melanin. Tricyclazole prevented melanin formation in the wild-type T. basicola and caused the endogenous accumulation of the melanin metabolites scytalone, flaviolin, 3,3′-biflaviolin, and 2-hydroxyjuglone. Vermelone and 1,8-dihydroxynaphthalene, but not scytalone, were converted to natural appearing melanins by the tricyclazole-treated wild-type fungus. The primary site of inhibition by tricyclazole was the enzymatic reduction of 1,3,8-trihydroxynaphthalene to vermelone. These results indicate that melanin biosynthesis in T. basicola involves the stepwise conversion of scytalone to melanin as reported previously for Verticillium dahliae. Tricyclazole appears to be a useful and expedient tool for the study of melanin biosynthesis in various fungi that utilize pentaketide metabolites as intermediates.

Melanin biosynthesis and the metabolism of flaviolin and 2-hydroxyjuglone inWangiella dermatitidis

Melanin biosynthesis in the human pathogenWangiella dermatitidis was inhibited by tricyclazole, causing pentaketide melanin metabolites to accumulate in the cultures. One of these metabolites, scytalone, was racemic and thus different than the (+)-enantiomer fromVerticillium dahliae. An albino mutant ofW. dermatitidis metabolized scytalone to a pigment ultrastructurally identical to wild-type melanin. Cell-free homogenates of the wild type carried out typical reductive and dehydrative reactions with known melanin intermediates and the reductive reactions were inhibited by tricyclazole. Other reductive and dehydrative reactions that utilize flaviolin and 2-hydroxyjuglone were studied anaerobically with homogenates from both the wild type and the albino mutant. The homogenates converted flaviolin to 5-hydroxyscytalone and products identical to those obtained from 2-hydroxyjuglone. The albino, in culture, carried out the same reactions with 2-hydroxyjuglone but metabolized flaviolin to a number of unknown colored products apparently through oxidative reactions. Similarities between the melanin pathway and the flaviolin and 2-hydroxyjuglone branch pathways are discussed and tricyclazole is shown to inhibit reductive reactions with naphthols in the three pathways.

Melanin biosynthesis inVerticillium dahliae: Dehydration and reduction reactions in cell-free homogenates

Abstract Cell-free homogenates and anaerobic conditions were used to study melanin biosynthesis in the imperfect fungusVerticillium dahliae Kleb. The homogenates reduced 1,3,6,8-tetrahydroxynaphthalene to scytalone and 1,3,8-trihydroxynaphthalene to vermelone. 1,3,6,8-Tetrahydroxynapthalene has not been isolated from fungi and was previously considered a hypothetical intermediate in the melanin pathway. This is the first report of its use as an exogenous melanin substrate. The enzymatic reductions required NADPH as a cofactor. The reactions were inhibited by the systemic fungicides tricyclazole (EL-291), pyroquilon (CGA-49104) and 4,5-dihydro-4-methyltetrazolo [1,5α]quinazolin-5-one (PP-389), and were eliminated by heating homogenates to 40°C for 35 minutes prior to the addition of substrate. The homogenates also enzymatically dehydrated scytalone to 1,3,8-trihydroxynaphthalene and vermelone to 1,8-dihydroxynaphthalene. The dehydration reactions did not require pyridine nucleotides, were insensitive to the systemic fungicides, and were eliminated after heating at 70°C for 35 minutes.

EVIDENCE FOR PENTAKETIDE MELANIN BIOSYNTHESIS IN DEMATIACEOUS HUMAN PATHOGENIC FUNGI

A wide variety of human pathogenic fungi of the form-family Dematiaceae are characterized by the production of dark brown to black vegetative hyphae. Recently it was reported that one of these fungi, Wangiella dermatitidis 8656 (Kano) McGinnis [=Exophila mansonii 8656 (Kano) DeHoog], a causative agent of phaeohyphomycosis, produces melanin via the pentaketide pathway (2, 8). The melanin was shown to be deposited in the outer cell walls of the fungus (8). Tricyclazole (5-methyl,2,4-triazolo[3,4b] benzothiazole) blocked the biosynthesis of melanin in W. dermatitidis (2) causing colored pentaketide metabolites to be secreted into the medium around fungal colonies. Chromatographic and spectrophotometric assays of the metabolites enabled the identification of three

Evidence for 1,8‐dihydroxynaphthalene melanin in three halophilic black yeasts grown under saline and non‐saline conditions

The ascomycetous black yeasts Hortaea werneckii, Phaeotheca triangularis, and Trimmatostroma salinum are halophilic fungi that inhabit hypersaline water of solar salterns. They are characterized by slow, meristematic growth and very thick, darkly pigmented cell walls. The dark pigment, generally thought to be melanin, is consistently present in their cell walls when they grow under saline and non-saline conditions. We used the inhibitor tricyclazole to test the fungi in this study for the presence of 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis, since fungal melanins reportedly are derived either from DHN, tyrosine via 3,4-dihydroxyphenylalanine, gamma-glutaminyl-3,4-dihydroxybenzene, or catechol. Tricyclazole-treated cultures of the fungi were reddish-brown in color and contained typical intermediates of the DHN-melanin pathway, as demonstrated by high-performance liquid chromatography. This investigation showed that the three fungi synthesized DHN-melanin under saline and non-saline growth conditions.

New Biosynthetic Step in the Melanin Pathway of Wangiella (Exophiala) dermatitidis: Evidence for 2-Acetyl-1,3,6,8-Tetrahydroxynaphthalene as a Novel Precursor

ABSTRACT The predominant cell wall melanin of Wangiella dermatitidis, a black fungal pathogen of humans, is synthesized from 1,8-dihydroxynaphthalene (D2HN). An early precursor, 1,3,6,8-tetrahydroxynaphthalene (T4HN), in the pathway leading to D2HN is reportedly produced directly as a pentaketide by an iterative type I polyketide synthase (PKS). In contrast, the bluish-green pigment in Aspergillus fumigatus is produced after the enzyme Ayg1p converts the PKS product, the heptaketide YWA1, to T4HN. Previously, we created a new melanin-deficient mutant of W. dermatitidis, WdBrm1, by random molecular insertion. From this strain, the altered gene WdYG1 was cloned by a marker rescue strategy and found to encode WdYg1p, an ortholog of Ayg1p. In the present study, two gene replacement mutants devoid of the complete WdYG1 gene were derived to eliminate the possibility that the phenotype of WdBrm1 was due to other mutations. Characterization of the new mutants showed that they were phenotypically identical to WdBrm1. Chemical analyses of mutant cultures demonstrated that melanin biosynthesis was blocked, resulting in the accumulation of 2-acetyl-1,3,6,8-tetrahydroxynaphthalene (AT4HN) and its oxidative product 3-acetylflaviolin in the culture media. When given to an albino W. dermatitidis strain with an inactivated WdPKS1 gene, AT4HN was mostly oxidized to 3-acetylflaviolin and deacetylated to flaviolin. Under reduced oxygen conditions, cell-free homogenates of the albino converted AT4HN to D2HN. This is the first report of evidence that the hexaketide AT4HN is a melanin precursor for T4HN in W. dermatitidis.

Isolation and characterisation of Gaeumannomyces graminis var. graminis melanin mutants

Gaeumannomyces graminis var. graminis produces 1,8-dihydroxynaphthalene (DHN) melanin in its hyphal and hyphopodial cell walls. We isolated G. graminis mutants that were affected in their melanin biosynthesis. One was unable to synthesize DHN-melanin and, because it accumulated 2-hydroxyjuglone, a DHN melanin pathway shunt product, it is most likely to be defective in the reductase that catalyzes the conversion of 1,3,8-trihydroxynaphthalene to vermelone, the penultimate reaction in DHN synthesis. Genetic crosses with our wild-type strain indicated that this trihydroxynaphthalene reductase deficiency was the result of a single mutation. Another mutant constitutively synthesized DHN melanin and genetic crosses with our wild-type strain suggested that this heavily melanized mutant had a single mutation responsible for its phenotype. This mutant produced more melanin than the wild-type strain as measured by Azure A binding to melanin. The wild type and constitutively melanized mutant hyphae were more hydrophobic and more resistant to lytic enzymes, benomyl, restrictive temperature, and uv light than the non-melanized mutant, which also autolysed more readily. The non-melanized mutant was not more sensitive to heavy metal than the melanized strains. In addition, the non-melanized mutant was unaltered in pathogenicity to rice, whereas the constitutively melanized mutant was less pathogenic. The constitutively melanized mutant produced less extracellular lytic enzymes than the wild-type and the non-melanized mutant, which may explain its reduced virulence.