| 1,8-Dihydroxynaphthalene (1,8-DHN) is a fungal polyketide that contributes to virulence when polymerized to DHN-melanin in the cell walls of Wangiella dermatitidis, an agent of phaeohyphomycoses in humans. To begin a genetic analysis of the initial synthetic steps leading to the production of DHN-melanin, a 772-bp PCR product was amplified from W. dermatitidis genomic DNA using primers based on conserved regions of fungal polyketide synthases (Pks) known to be responsible for the synthesis of the first cyclized DHN-melanin pathway intermediate, 1,3,6,8-tetrahydroxynaphthalene. The cloned PCR product was then used as a targeting sequence to disrupt the polyketide synthase gene (WdPKS1) in W. dermatitidis . The resulting wdpks1Delta disruption mutants 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, sequenced, and shown to encode a putative polyketide synthase (WdPks). This identification was substantiated by finding highly conserved Pks domains for a beta-ketoacyl synthase domain, an acetyl/malonyl transferase domain, two acyl carrier protein (ACP) domains and a thioesterase domain in a single open reading frame consisting of three exons separated by two short introns. Following the production of additional wdpks1Delta mutants, studies using an acute mouse model confirmed that all wdpks1 Delta strains were less virulent, and that the reconstitution of DHN-melanin biosynthesis in wdpks1Delta-1 reestablished its ability to cause fatal infections. A combination of flow cytometry and a colony-count-dependent method assay of the wild-type strain, a wdpks1Delta disruption mutant strain and wdpks1Delta-1 complemented with WdPKS1 also demonstrated that melanin prevented this pathogen from being killed in the phagolysome of neutrophils. However, the melanin did not influence phagocytosis by or the oxidative burst of the neutrophils involved. I suspect that the ability of W. dermatitidis melanin to block the effects of the neutrophil oxidative burst critically impairs the potential of a host to eliminate this fungal pathogen by normal immunological processes and thus plays an important role in the pathogenesis in phaeohyphomycosis. (Abstract shortened by UMI.)... |
