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Aspergillus fumigatus is the most prevalent airborne filamentous fungal pathogen in humans, causing severe and often fatal invasive infections in immunocompromised patients. Currently available antifungal drugs to treat invasive aspergillosis have limited modes of action, and few are safe and effective. To identify and prioritize antifungal drug targets, we have developed a conditional promoter replacement (CPR) strategy using the nitrogen-regulated A. fumigatus NiiA promoter (pNiiA). The gene essentiality for 35 A. fumigatus genes was directly demonstrated by this pNiiA-CPR strategy from a set of 54 genes representing broad biological functions whose orthologs are confirmed to be essential for growth in Candida albicans and Saccharomyces cerevisiae. Extending this approach, we show that the ERG11 gene family (ERG11A and ERG11B) is essential in A. fumigatus despite neither member being essential individually. In addition, we demonstrate the pNiiA-CPR strategy is suitable for in vivo phenotypic analyses, as a number of conditional mutants, including an ERG11 double mutant (erg11BΔ, pNiiA-ERG11A), failed to establish a terminal infection in an immunocompromised mouse model of systemic aspergillosis. Collectively, the pNiiA-CPR strategy enables a rapid and reliable means to directly identify, phenotypically characterize, and facilitate target-based whole cell assays to screen A. fumigatus essential genes for cognate antifungal inhibitors.

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...

Candida albicans is not inhibited by a number of drugs known to affect fungal cells. The basis for this resistance in most cases is unknown but has been attributed to the general impermeability of the fungal cell envelope. A gene (BENr) formerly shown to be responsible for the resistance of C. albicans to benomyl and methotrexate was shown in the present study to confer resistance to four other inhibitory compounds: cycloheximide, benztriazoles, 4-nitroquinoline-N-oxide, and sulfometuron methyl. Analysis of the protein database revealed an apparent similarity of the C. albicans gene to membrane protein genes encoding antibiotic resistance in prokaryotes and eukaryotes and a high degree of identity to a recently cloned gene encoding cycloheximide resistance in Candida maltosa. We propose that BENr encodes a protein that operates in a fashion similar, but not identical, to that described for other multiple-drug resistance proteins.

The use of poly(lactide)-based materials is, in part, limited by their physical and mechanical properties. This article reviews the methods that have been employed to enable enhancement of the materials properties through synthetic manipulation of the polymer structure including block copolymer synthesis and modification of the lactide monomer structure, focusing on the application of ring-opening polymerization. In turn the effect of these structural modifications on the properties of the resultant materials are reported.