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 (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.

Figure 1. Outline of the A. fumigatus pNiiA-CPR Strategy

(A) A cloning vector, pPyrG-pNiiA, was created specifically for the construction of CPR cassettes. This plasmid contains an A. niger pyrG (encoding orotidine-5′-phosphate decarboxylase and required for uridine-uracil prototrophy) as a selection marker [41], as well as an Amp⁺ selection marker and the A. fumigatus pNiiA conditional promoter. Unique restriction sites have been engineered at either side of the pyrG-pNiiA cassette to facilitate subcloning of flanking sequences. (B) Schematic overview of the pNiiA-CPR strategy and strain confirmation by PCR genotyping. Promoter replacement cassettes were constructed by inserting approximately 1.5 kb of homologous flanking sequences of the target gene (L-arm and R-arm) into the Not1/Mlu1 and Asc1/Pac1 restriction sites, respectively. After Not1 and Pac1 double digestion, the linearized pNiiA-CPR cassette was introduced into CEA17 strain (pyrG⁻) by protoplast transformation. As indicated, three sets of primers were used to perform genotypic PCRs to map the expected promoter replacement junctions (L1/L2 for left-arm junction, R1/R2 for right-arm junction) and to confirm the deletion of the native promoter (L1/P). (C) Phenotype of transformants obtained by the pNiiA-CPR strategy for MET2, TUB1, GFA1, and ALR1. CPR mutants were examined under pNiiA-inducing (AMM plus nitrate) and repressing (AMM plus ammonium) conditions after 36 h at 30 °C. (D) Growth phenotypes of GFA1, PFS2, ALG7, CDC24, and ALR1 pNiiA-CPR mutants under these standard repressing conditions are scored qualitatively as the following: 4+, essential for cell viability, no growth under repressing conditions; 3.5+ or 3+, showing very strong or strong growth defect; 2+ or 1+, mild to minor growth defect; and 0+, no growth phenotype observed.

Figure 2. Genetic Evaluation of the pNiiA-CPR Strategy

(A) Phenotypes of pNiiA-CPR mutants for HIS3, TRP5, MET16, MET2, LYS9, and LYS4. Under standard repressing conditions (Re, AMM plus ammonium), all strains lacked detectable growth (4+ phenotype). Growth was unimpaired under inducing conditions (In; AMM plus nitrate). Growth phenotypes of pNiiA-CPR mutants under repressing conditions were specifically suppressed if the cognate amino acid was provided to the growth media (His, histidine; Trp, tryptophan; Met, methionine; or Lys, lysine). (B) Growth phenotypes of pNiiA-CPR mutants for the previously reported A. fumigatus essential genes, FKS1, GUS1, SPE2, and HEM15 [12,13]. Reproducible 4+ essential growth phenotypes are observed for each pNiiA-CPR mutant under repressing conditions with the exception of FKS1, which produced a 3.5+ growth phenotype. (C) arp2 and ayg1 conidial color phenotypes by the pNiiA-CPR strategy. Wild-type (WT) strain CEA10, pNiiA-arp2, and pNiiA-ayg1 mutants display normal dark-green conidia color under inducing conditions. Gene-specific conidia color phenotypes characteristic of their known null phenotype [19] are specifically detected under repressing conditions. (D) nudC growth phenotype by the pNiiA-CPR strategy. Highly reproducible growth and morphological phenotypes associated with nudC mutants are observed (see Figure S1) as similarly determined using an alcA heterologous conditional promoter [15].

Figure 3. Expressional Level Analysis of pNiiA-CPR Mutants

(A) RT-PCR of pNiiA-MET2: (a) pNiiA-MET2 mutant under inducing conditions, (b) pNiiA-MET2 mutant under repressing conditions plus 100 μg/ml methionine, (c) pNiiA-MET2 mutant under inducing condition plus 100 μg/ml methionine, and (d) wild-type A. fumigatus strain (CEA10) under repressing conditions. To monitor and ensure even sample loading, RT-PCRs for the ACT1 transcript were also performed using identical samples. In addition, standard PCR was performed to confirm that there is no detectable genomic-DNA contamination. (B) Northern blot analysis of pNiiA-ALR1 mutant expression levels. Northern blot was performed with RNA samples prepared from the nonessential ALR1 mutant and wild-type cells growing in standard inducing (In) or repressing (Re) conditions. ACT1 transcript levels served as sample loading controls. (C) Real-time RT-PCR analysis of expression level of pNiiA-ALR1 and pNiiA-MET2. pNiiA-CPR mutants and wild-type strain (CEA10) were grown in inducing (In) or repressing (Re) medium at 37 °C for 20 h, and total RNA was extracted from identical time points. The relative expression level normalized to total input RNA [43] is displayed on the y-axis. Error bars represent SD. Compared to wild-type, the relative expression level for ALR1 and MET2 is 0.013 and 0.061 under repressing conditions and 2.49 and 23.58 under inducing conditions, respectively. (D) pNiiA-TUB1 expression level under inducing (In) and repressing (Re) conditions versus wild-type level is displayed on the y-axis. Error bar represents SD. The relative expression level of pNiiA-TUB1 versus wild-type is 0.06 under repressing conditions and 6.35 under inducing conditions, respectively. Note: Since real-time RT-PCR was performed using primers detecting both pNiiA-TUB1 and wild-type allele, data shown were calculated by subtracting wild-type level from total inducing (In) and total repressing (Re) level, respectively.

Figure 4. Analysis of pNiiA-CPR Associated Morphological Terminal Phenotypes

Terminal growth phenotypes of pNiiA-CPR mutants were observed under a microscope (×160) with conidia grown for 36 to 40 h at 30 °C under standard repressing conditions. A continuum of conidia germination phenotypes of high penetrance was observed; ranging from those completely failing to undergo polarized growth (SEC31, SLY1) or swollen and highly disorganized condidia (GFA1), to those displaying stunted (TUB1, ERG10) or nonbranching germlings with swollen conidia (HEM15) with only rudimentary polarized growth. Micromycelial colonies were observed for a pNiiA-FKS1 mutant and resembling the morphology of wild-type A. fumigatus when grown in the presence of minimum effective concentration (MEC) of the FKS1p inhibitor, caspofungin [21]. Growth phenotypes under inducing conditions are shown in Figure S3.

Figure 5. Determination of Cidal or Static Terminal Phenotypes

A representative example of cidal and static terminal phenotypes for GFA1 and TRR1 pNiiA-CPR mutants is shown. (A) pNiiA-GFA1 displayed a cidal terminal phenotype as a dramatic (greater than 90%) reduction in CFU was observed after incubation in pNiiA-repressing conditions for 24 or 48 h. (B) pNiiA-TRR1 revealed a static terminal phenotype as no significant reduction in CFU counts was detected after 48-h incubation under repressing conditions. A summary of all additional cidal/static terminal phenotypes for A. fumigatus genes displaying 4+ qualitative growth phenotypes is provided (see Table 2).

Figure 6. Phenotypic Analyses of ERG11 Gene Family

Growth phenotypes of ERG11 gene family were observed with pNiiA-CPR mutants, null deletion mutants, and double mutants (erg11BΔ, pNiiA-ERG11A). Strains were grown on either inducing medium (AMM plus nitrate) or repressing medium (AMM plus ammonium) at 30 °C for 40 h.

Figure 7. In Vivo Validation of A. fumigatus pNiiA-CPR Mutants in an Immunocompromised Murine Model of Systemic Infection

(A) pNiiA-CPR mutants growing on AMM plus 20% mouse serum (Sigma) reproduced their terminal growth phenotypes as observed on either AMM plus ammonium or rich medium (Table 2). (B and C) In vivo validation of pNiiA-CPR mutants. (B) ICR male mice were immunocompromised by administration of cyclophosphamide at 150 mg/kg twice prior to infection and 100 mg/kg twice a week after infection. Approximately 10⁵ viable conidia from individual pNiiA-CPR mutants (TUB1, SEC31, GCD6, GFA1, MET2, and AUR1) were injected into the tail vein of immunocompromised mice (five mice per group). Signs of infection were monitored for up to 12 d following infection. Wild-type strain CEA10 and the starting strain CEA17 (a pyrG ⁻ auxotroph of CEA10) [25,39] were included as positive controls for virulence and avirulence, respectively. (C) Genetic inactivation of the ERG11 gene family promotes avirulence in an immunocompromised murine model of systemic infection. Pathogenesis of erg11AΔ, erg11BΔ, and an ERG11 double mutant (erg11BΔ, pNiiA-ERG11A) was similarly analyzed (as described above) but over a longer postinfection period (22 d), and animal survival was compared to CEA10 and CEA17 control strains.