Molecular Cloning and Characterization of WdPKS1 , a Gene Involved in Dihydroxynaphthalene Melanin Biosynthesis and Virulence in Wangiella ( Exophiala ) dermatitidis

Abstract

1,8-Dihydroxynaphthalene (1,8-DHN) is a fungal polyketide that contributes to virulence when polymerized to 1,8-DHN melanin in the cell walls of Wangiella dermatitidis, an agent of phaeohyphomycosis in humans.To begin a genetic analysis of the initial synthetic steps leading to 1,8-DHN melanin biosynthesis, a 772-bp PCR product was amplified from genomic DNA using primers based on conserved regions of fungal polyketide synthases (Pks) known to produce the first cyclized 1,8-DHN-melanin pathway intermediate, 1,3,6,8-tetrahydroxynaphthalene. The cloned PCR product was then used as a targeting sequence to disrupt the putative polyketide synthase gene, WdPKS1, in W. dermatitidis. The resulting wdpks1⌬ disruptants showed no morphological defects other than an albino phenotype and grew at the same rate as their black wild-type parent. Using a marker rescue approach, the intact WdPKS1 gene was then successfully recovered from two plasmids. The WdPKS1 gene was also isolated independently by complementation of the mel3 mutation in an albino mutant of W. dermatitidis using a cosmid library. Sequence analysis substantiated that WdPKS1 encoded a putative polyketide synthase (WdPks1p) in a single open reading frame consisting of three exons separated by two short introns. This conclusion was supported by the identification of highly conserved Pks domains for a ␤-ketoacyl synthase, an acetyl-malonyl transferase, two acyl carrier proteins, and a thioesterase in the deduced amino acid sequence. Studies using a neutrophil killing assay and a mouse acute-infection model confirmed that all wdpks1⌬ strains were less resistant to killing and less virulent, respectively, than their wild-type parent. Reconstitution of 1,8-DHN melanin biosynthesis in a wdpks1⌬ strain reestablished its resistance to killing by neutrophils and its ability to cause fatal mouse infections.The zoopathogenic fungus Wangiella (Exophiala) dermatitidis is one of many form species of the Fungi Imperfecti, which are darkly pigmented (dematiaceous) owing to the deposition of 1,8-dihydroxynaphthalene (1,8-DHN) melanin in their cell walls (22,45). This fungus has recently become better known as a paradigm for the causative agents of phaeohyphomycosis and other emerging dermatomycoses of humans, because of its increasing detection as a systemic pathogen in both immunocompetent and immunocompromised patients (34, 35). Moreover, because W. dermatitidis has a well-defined polymorphic nature and a well-characterized cell wall chemistry, it serves as an excellent model for the more than 100 other dematiaceous fungal pathogens of humans (14,37,42).Although dark pigments of fungi are often called melanin without regard to mode of enzymatic synthesis or chemical composition, most syntheses of melanin are attributed to either a phenoloxidase, e.g., laccases and tyrosinases, or a polyketide synthase (Pks) of a pentaketide biosynthetic pathway (52). The phenoloxidases have been found mostly among basidiomycete fungi and are usually composed of soluble enzymes with broad substra...

A PKS gene, pks-1, is involved in chaetoglobosin biosynthesis, pigmentation and sporulation in Chaetomium globosum

“…From these data, we conclude that pks-1 is, in part, responsible for melanin biosynthesis in C. globosum. The gene pks-1 encodes a polypeptide with 2181 amino acids which harbors five domains that are found in other fungal polyketide synthases ( Figure 1A), suggesting that melanin biosynthesis in C. globosum is accomplished via the DHN pathway defined in various fungi [22,47,48]. To confirm this, tricyclazole, an inhibitor of the DHN pathway, was added to plates.…”

WdChs1p, a class II chitin synthase, is more responsible than WdChs2p (Class I) for normal yeast reproductive growth in the polymorphic, pathogenic fungus Wangiella (Exophiala) dermatitidis

“…Although traditionally most associated with primary dermatotrophic forms of cutaneous and subcutaneous phaeohyphomycosis, this asexual fungus has recently become better known as a paradigm for this emerging mycosis because of its increasing detection as a systemic pathogen in both immunocompetent and immunocompromised patients (Matsumoto et al 1993(Matsumoto et al , 1994manipulated polymorphic vegetative nature, a well-characterized cell wall chemistry and a rapidly improving molecular tractability, it currently serves as the model of choice for discovering cell wall-related virulence determinants among the dematiaceous fungi, irrespective of the mycoses they cause and the morphotypes of the agents involved (Szaniszlo et al 1983;Montijin et al 1997;Kwon-Chung et al 1998;Szaniszlo 2002). The model studies have confirmed by molecular methods that both 1,8-dihydroxynaphthlene (1,8-DHN) melanin and chitin, a linear homopolymer of β(1 → 4) N-acetylglucosamine, have relevance to the virulence of W. dermatitidis, and therefore presumably the virulence of other dematiaceous pathogens of humans (Feng et al 2001;Wang et al 2001;Szaniszlo 2002;Liu et al 2004). They have also shown that chitin and β(1 → 3) glucan serve as the main structural constituents of the cell walls of W. dermatitidis and that chitin content varies widely with morphotype (Szaniszlo et al 1983;Montijin et al 1997).…”

The genome sequence of the filamentous fungus Neurospora crassa

“…6). The first class contains genes similar to DHN-melanin PKS genes of the fungi Exophila dermatitidis 61 , Colletotrichum lagenarium 62 and Alternaria alternata 63 . Sequence identity to numerous expressed sequence tag (EST) sequences from sexual and perithecial libraries suggest a role in melanin pigment synthesis during sexual development 64 .…”