Essential gene identification and drug target prioritization in Aspergillus fumigatus.
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 (erg11BDelta, 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.
Hu
Wenqi
W
Merck Frosst Center of Fungal Genetics, Montreal, Quebec, Canada.
Sillaots
Susan
S
Lemieux
Sebastien
S
Davison
John
J
Kauffman
Sarah
S
Breton
Anouk
A
Linteau
Annie
A
Xin
Chunlin
C
Bowman
Joel
J
Becker
Jeff
J
Jiang
Bo
B
Roemer
Terry
T
D000818:Animals; D000935:Antifungal Agents / Q000627:therapeutic use; D001228:Aspergillosis / Q000382:microbiology*; D001232:Aspergillus fumigatus / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D004195:Disease Models, Animal; D016503:Drug Delivery Systems; D015966:Gene Expression Regulation, Fungal; D020043:Genes, Essential; D005800:Genes, Fungal; D008297:Male; D051379:Mice; D008819:Mice, Nude; D008969:Molecular Sequence Data; D009565:Nitrate Reductases / Q000235:genetics; D010088:Oxidoreductases / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D011995:Recombination, Genetic; D058886:Sterol 14-Demethylase; D014158:Transcription, Genetic; D014774:Virulence / Q000235:genetics / Q000502:physiology
D000935:Antifungal Agents; C548418:CYP51A1 protein, human; D004271:DNA, Fungal; D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; C548415:Cyp51 protein, mouse; D058886:Sterol 14-Demethylase; D009565:Nitrate Reductases