Targeted Disruption of Melanin Biosynthesis Genes in the Human Pathogenic Fungus Lomentospora prolificans and Its Consequences for Pathogen Survival.

Ayat Al-Laaeiby,Christopher Thornton,Michael Kershaw,Tina Penn

doi:10.3390/ijms17040444

Ayat Al-Laaeiby, Christopher Thornton + Show 2 more

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https://doi.org/10.3390/ijms17040444

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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.

Highlights

Lomentospora prolificans is a dematiaceous fungus that has emerged over recent years as a serious and often life-threatening pathogen of immunocompromised individuals including those with AIDS, haematological malignancies and bone marrow and solid organ transplants [1,2,3,4,5,6,7,8]
Homologous recombination resulted in the replacement of the SCD1 open reading frame (ORF) with the functional hygromycin B phosphotransferase (HPH) gene
The pigment melanin is an integral component of the fungal cell wall conferring protection to stresses that involve cell damage such as UV radiation and reactive oxygen species [22,25,26,27,28,29,30,32,33] and which has been shown in other human pathogenic fungi to contribute to amphotericin B resistance [24,25,41]

Summary

Introduction

Lomentospora prolificans (formally Scedosporium prolificans) is a dematiaceous (melanised) fungus that has emerged over recent years as a serious and often life-threatening pathogen of immunocompromised individuals including those with AIDS, haematological malignancies and bone marrow and solid organ transplants [1,2,3,4,5,6,7,8]. Deep-seated infections disseminate rapidly with associated mortality rates of up to 80% [1,9]. Infections due to near-drowning were reported by the World Health Organisation after the Great Eastern Japan earthquake and tsunami in 2011 [10,11]. 2016, 17, 444 tsunami in 2011 [10,11]. Recovery of L. prolificans from the sputum of cystic fibrosis patients has been reported, but disease exacerbation due to the fungus has yet to be established [12]

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