Effects of disrupting the polyketide synthase gene WdPKS1 in Wangiella [Exophiala] dermatitidis on melanin production and resistance to killing by antifungal compounds, enzymatic degradation, and extremes in temperature.
BACKGROUND
Wangiella dermatitidis is a human pathogenic fungus that is an etiologic agent of phaeohyphomycosis. W. dermatitidis produces a black pigment that has been identified as a dihydroxynaphthalene melanin and the production of this pigment is associated with its virulence. Cell wall pigmentation in W. dermatitidis depends on the WdPKS1 gene, which encodes a polyketide synthase required for generating the key precursor for dihydroxynaphthalene melanin biosynthesis.


RESULTS
We analyzed the effects of disrupting WdPKS1 on dihydroxynaphthalene melanin production and resistance to antifungal compounds. Transmission electron microscopy revealed that wdpks1Delta-1 yeast had thinner cell walls that lacked an electron-opaque layer compared to wild-type cells. However, digestion of the wdpks1Delta-1 yeast revealed small black particles that were consistent with a melanin-like compound, because they were acid-resistant, reacted with melanin-binding antibody, and demonstrated a free radical signature by electron spin resonance analysis. Despite lacking the WdPKS1 gene, the mutant yeast were capable of catalyzing the formation of melanin from L-3,4-dihyroxyphenylalanine. The wdpks1Delta-1 cells were significantly more susceptible to killing by voriconazole, amphotericin B, NP-1 [a microbicidal peptide], heat and cold, and lysing enzymes than the heavily melanized parental or complemented strains.


CONCLUSIONS
In summary, W. dermatitidis makes WdPKS-dependent and -independent melanins, and the WdPKS1-dependent deposition of melanin in the cell wall confers protection against antifungal agents and environmental stresses. The biological role of the WdPKS-independent melanin remains unclear.
Paolo
William F
WF
Department of Medicine, Albert Einstein College of Medicine, Bronx, NY, USA. paolow01@med.nyu.edu
Dadachova
Ekaterina
E
Mandal
Piyali
P
Casadevall
Arturo
A
Szaniszlo
Paul J
PJ
Nosanchuk
Joshua D
JD
D000935:Antifungal Agents / Q000494:pharmacology*; D003080:Cold Temperature; D004305:Dose-Response Relationship, Drug; D025141:Drug Resistance, Fungal / Q000235:genetics; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004795:Enzyme Stability / Q000235:genetics; D005093:Exophiala / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D006358:Hot Temperature; D008543:Melanins / Q000096:biosynthesis*; D008825:Miconazole / Q000494:pharmacology; D008855:Microscopy, Electron, Scanning / Q000379:methods; D046529:Microscopy, Electron, Transmission / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods; D009154:Mutation / Q000235:genetics; D009284:Naphthols; D048630:Polyketide Synthases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C423455:1,8-dihydroxynaphthalene melanin; D000935:Antifungal Agents; D008543:Melanins; D009284:Naphthols; D048630:Polyketide Synthases; D008825:Miconazole