Essential Gene Identification and Drug Target Prioritization in Aspergillus fumigatus

Wenqi Hu,Sebastien Lemieux,Joel Bowman,John Davison,Annie Linteau,Jeff Becker,Chunlin Xin,Susan Sillaots,Anouk Breton,Terry Roemer,Bo Jiang,Sarah Kauffman,Brendan P Cormack

doi:10.1371/journal.ppat.0030024

Wenqi Hu, Sebastien Lemieux + Show 11 more

Open Access

https://doi.org/10.1371/journal.ppat.0030024

Copy DOI

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.

Highlights

Aspergillus fumigatus is a ubiquitous soil-dwelling saprophytic fungus that propagates through the prolific production of air-borne conidia [1]
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 and pathogenesis in a murine model of systemic aspergillosis to prioritize essential genes as novel drug targets suitable for developing broad-spectrum antifungal agents

Summary

Introduction

Aspergillus fumigatus is a ubiquitous soil-dwelling saprophytic fungus that propagates through the prolific production of air-borne conidia [1]. It has been recently suggested that A. fumigatus pathogenesis is based on its saprophytic lifestyle in combination with the immunosuppressed state of the host, rather than from genuine fungal virulence factors [2]. Current treatment options for invasive aspergillosis are limited to three classes of antifungal therapeutics: polyenes (amphotericin B and various liposomal formulations), azoles (e.g., fluconazole, voriconazole, itraconazole), and, more recently, semisynthetic echinocandins (e.g., caspofungin and anidulafungin) [4,5]. Mortality associated with invasive aspergillosis remains high (ranging from 60% to 90%) and more efficacious antifungal drugs with novel mechanisms of action are needed [4,5]

Methods

Results

Discussion

Conclusion