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. Melanin was shown to play no role in the protection of the pathogen from immune cell recognition and killing by alveolar macrophages, with similar degrees of engulfment, and spore viabilities, of mutant and wild-type strains after phagocytosis. Contrary to expectations, the albino PKS-deficient mutant was significantly more virulent than the melanised wild-type strain during pathogenicity studies in the invertebrate infection model Galleria mellonella, with levels of virulence restored to near wild-type levels in the complemented strain ΔLppks1::hph:PKS. Taken together, the results presented in this thesis show that melanin protects L. prolificans from UV radiation and from oxidative killing by H2O2, consistent with its survival in extreme environmental habitats. However, melanin was 3 not found to play a role in the resistance of the pathogen to the antifungal drug amphotericin B, to protect the fungus from immune cell recognition or killing by alveolar macrophages, or to its pathogenicity.

>Magister Scientiae - MSc / Tyrosinase is an enzyme widely distributed in the biosphere and is found in many species of bacteria, fungi, animals, and plants; it is associated with melanin production. Even though it possesses many beneficial properties such as photoprotection, but overproduction causes undesirable effects such melasma, solar lentigines etc. Therefore, tyrosinase enzyme inhibitors are of far-ranging importance in cosmetics, medicinal products, and food industries. This study is aimed to test anti-tyrosinase activity in 37 plants from 20 families using mushroom tyrosinase inhibition method; each plant was extracted with methanol. The results showed that 17 plant extracts, exerted a considerable level of in vitro tyrosinase inhibition comparable to positive controls of kojic acid in the same solvent systems when evaluated spectrophotometrically. Among plant extracts, those that showed an inhibition rate >50 % at 50 μg/ml and ˃60 % at 200 μg/ml were A. karroo (Hayne.), A. afra Jacq. Ex Willd, C. geifolia (L.), E. racemosa (L.), H. petiolare Hilliard & B.L.Burt, M. quercifolia (L.), M. communis (L.), P. rigida (Wikstr.), P. ecklonii (Benth.), P. ericoides (L.), S. Africanacaerulea (L.), S. Africana-lutea (L.), S. antarcticus (Willd.), S. lucida (L.) F.A.Barkley, S. hamilifolius (L.), S. furcellata R.Br and T riparia which exhibited great anti-tyrosinase activity.