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The frequency of antifungal resistance, particularly to the azole class of ergosterol biosynthetic inhibitors, is a growing global health problem. Survival rates for those infected with resistant isolates are exceptionally low. Beyond modification of the drug target, our understanding of the molecular basis of azole resistance in the fungal pathogen Aspergillus fumigatus is limited. We reasoned that clinically relevant antifungal resistance could derive from transcriptional rewiring, promoting drug resistance without concomitant reductions in pathogenicity. Here we report a genome-wide annotation of transcriptional regulators in A. fumigatus and construction of a library of 484 transcription factor null mutants. We identify 12 regulators that have a demonstrable role in itraconazole susceptibility and show that loss of the negative cofactor 2 complex leads to resistance, not only to the azoles but also the salvage therapeutics amphotericin B and terbinafine without significantly affecting pathogenicity. Resistance to primary treatments of invasive aspergillosis is growing. Here, the authors generate a knockout library for 484 transcription factors in Aspergillus fumigatus, and show that loss of the NCT complex leads to cross-resistance to all primary and some salvage therapeutics without affecting pathogenicity. [ABSTRACT FROM AUTHOR]

PCR product-based gene disruption has greatly accelerated molecular analysis of Saccharomyces cerevisiae. This approach involves amplification of a marker gene (e.g., URA3) including its flanking regulatory (promoter and polyadenylation) regions using primers that include at their 5′ ends about 50 bases of homology to the targeted gene. Unfortunately, this approach has proved less useful in organisms with higher rates of non-homologous recombination; e.g., in the yeast Candida glabrata, desired recombinants represent ≤2% of transformants. We modified the PCR-based approach by eliminating marker-flanking regions and precisely targeting recombination such that marker expression depends on the regulatory sequences of the disrupted gene. Application of this promoter-dependent disruption of genes (PRODIGE) method to three C. glabrata genes ( SLT2, LEM3, and PDR1) yielded desired recombinants at frequencies of 20, 31, and 11%, the latter representing a weakly expressed gene. For Candida albicans LEM3 and RHO1, specificity was 79–95% for one or both alleles, >sixfold higher than the published results with conventional PCR-based gene disruption. All 5 C. glabrata and C. albicans mutants had predicted phenotypes of calcofluor hypersensitivity ( slt2Δ and RHO1/rho1Δ), cycloheximide hypersensitivity ( pdr1Δ), or miltefosine resistance ( lem3Δ and lem3Δ/ lem3Δ). PRODIGE application to the S. cerevisiae PDR5 gene in strains with and without the Pdr1–Pdr3 transcriptional activators of this gene confirmed that transformant yield and growth rate depend on promoter strength. Using this PDR5 promoter- URA3 recombinant, we further demonstrate a simple extension of the method that yields regulatory mutants via 5-fluoroorotic acid selection. PRODIGE warrants testing in other yeast, molds, and beyond. [ABSTRACT FROM AUTHOR]