Essential Gene Identification and Drug Target Prioritization in Aspergillus fumigatus

Abstract

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. Aspergillus fumigatus is an opportunistic filamentous fungal pathogen of emerging clinical significance. Although virulence factors are seen as potential drug targets, neither genetic analyses nor genomic comparisons have identified genuine virulence factors in A. fumigatus. Essential genes required for fungal growth and viability also serve as potential drug targets, yet few have been described in this pathogen. To begin to catalog essential genes in A. fumigatus, we devised a genetic strategy for creating conditional mutants by promoter replacement of target genes using a nitrogen-regulated promoter. Applying this genetic approach to A. fumigatus genes orthologous to known essential genes of the nonpathogenic yeast, Saccharomyces cerevisiae and Candida albicans, we demonstrate a robust enrichment for identifying essential genes conserved within this pathogen. We show that A. fumigatus conditional mutants can be evaluated according to their terminal phenotypes (e.g., conidial germination, growth, morphology, and cidal versus static consequences) and pathogenesis in a murine model of systemic aspergillosis to prioritize essential genes as novel drug targets suitable for developing broad-spectrum antifungal agents.