title: Targeted Disruption of Melanin Biosynthesis Genes in the Human Pathogenic Fungus Lomentospora prolificans and Its Consequences for Pathogen Survival.
abstract: The dematiaceous (melanised) fungus Lomentospora (Scedosporium) prolificans is a life-threatening opportunistic pathogen of immunocompromised humans, resistant to anti-fungal drugs. Melanin has been shown to protect human pathogenic fungi against antifungal drugs, oxidative killing and environmental stresses. To determine the protective role of melanin in L. prolificans to oxidative killing (H₂O₂), UV radiation and the polyene anti-fungal drug amphotericin B, targeted gene disruption was used to generate mutants of the pathogen lacking the dihydroxynaphthalene (DHN)-melanin biosynthetic enzymes polyketide synthase (PKS1), tetrahydroxynapthalene reductase (4HNR) and scytalone dehydratase (SCD1). Infectious propagules (spores) of the wild-type strain 3.1 were black/brown, whereas spores of the PKS-deficient mutant ΔLppks1::hph were white. Complementation of the albino mutant ΔLppks1::hph restored the black-brown spore pigmentation, while the 4HNR-deficient mutant ΔLp4hnr::hph and SCD-deficient mutant ΔLpscd1::hph both produced orange-yellow spores. The mutants ΔLppks1::hph and ΔLp4hnr::hph showed significant reductions in spore survival following H₂O₂ treatment, while spores of ΔLpscd1::hph and the ΔLppks1::hph complemented strain ΔLppks1::hph:PKS showed spore survivals similar to strain 3.1. Spores of the mutants ΔLp4hnr::hph and ΔLpscd1::hph and complemented strain ΔLppks1::hph:PKS showed spore survivals similar to 3.1 following exposure to UV radiation, but survival of ΔLppks1::hph spores was significantly reduced compared to the wild-type strain. Strain 3.1 and mutants ΔLp4hnr::hph and ΔLppks1::hph:PKS were resistant to amphotericin B while, paradoxically, the PKS1- and SCD1-deficient mutants showed significant increases in growth in the presence of the antifungal drug. Taken together, these results show that while melanin plays a protective role in the survival of the pathogen to oxidative killing and UV radiation, melanin does not contribute to its resistance to amphotericin B.
authors.affiliation: Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK. aiea201@exeter.ac.uk.|Cell and Biotechnology Research Unit, College of Science, University of Basrah, Basrah 61004, Iraq. aiea201@exeter.ac.uk.
authors.forename: Ayat
authors.initials: A
authors.lastname: Al-Laaeiby
authors.affiliation: Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK. M.J.Kershaw@exeter.ac.uk.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Kershaw
authors.affiliation: Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK. T.J.Penn@exeter.ac.uk.
authors.forename: Tina J
authors.initials: TJ
authors.lastname: Penn
authors.affiliation: Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter EX4 4QD, UK. C.R.Thornton@ex.ac.uk.
authors.forename: Christopher R
authors.initials: CR
authors.lastname: Thornton
mesh_terms: D000666:Amphotericin B / Q000494:pharmacology; D000935:Antifungal Agents / Q000494:pharmacology; D015139:Blotting, Southern; D005656:Fungal Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D005658:Fungi / Q000187:drug effects / Q000378:metabolism* / Q000528:radiation effects; D006801:Humans; D006836:Hydro-Lyases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000633:toxicity; D008543:Melanins / Q000096:biosynthesis*; D048630:Polyketide Synthases / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014466:Ultraviolet Rays
chemical_list: D000935:Antifungal Agents; D005656:Fungal Proteins; D008543:Melanins; D048630:Polyketide Synthases; D000666:Amphotericin B; D006861:Hydrogen Peroxide; D006836:Hydro-Lyases; C082741:scytalone dehydratase
keywords: Lomentospora; fungus; gene; melanin; pathogen; polyketide synthase; scytalone dehydratase; tetrahydroxynaphthalene reductase