Reverse genetics in Candida albicans predicts ARF cycling is essential for drug resistance and virulence

Abstract

Candida albicans, the major fungal pathogen of humans, causes life-threatening infections in immunocompromised individuals. Due to limited available therapy options, this can frequently lead to therapy failure and emergence of drug resistance. To improve current treatment strategies, we have combined comprehensive chemical-genomic screening in Saccharomyces cerevisiae and validation in C. albicans with the goal of identifying compounds that can couple with the fungistatic drug fluconazole to make it fungicidal. Among the genes identified in the yeast screen, we found that only AGE3, which codes for an ADP-ribosylation factor GTPase activating effector protein, abrogates fluconazole tolerance in C. albicans. The age3 mutant was more sensitive to other sterols and cell wall inhibitors, including caspofungin. The deletion of AGE3 in drug resistant clinical isolates and in constitutively active calcineurin signaling mutants restored fluconazole sensitivity. We confirmed chemically the AGE3-dependent drug sensitivity by showing a potent fungicidal synergy between fluconazole and brefeldin A (an inhibitor of the guanine nucleotide exchange factor for ADP ribosylation factors) in wild type C. albicans as well as in drug resistant clinical isolates. Addition of calcineurin inhibitors to the fluconazole/brefeldin A combination only initially improved pathogen killing. Brefeldin A synergized with different drugs in non-albicans Candida species as well as Aspergillus fumigatus. Microarray studies showed that core transcriptional responses to two different drug classes are not significantly altered in age3 mutants. The therapeutic potential of inhibiting ARF activities was demonstrated by in vivo studies that showed age3 mutants are avirulent in wild type mice, attenuated in virulence in immunocompromised mice and that fluconazole treatment was significantly more efficacious when ARF signaling was genetically compromised. This work describes a new, widely conserved, broad-spectrum mechanism involved in fungal drug resistance and virulence and offers a potential route for single or improved combination therapies.

Figure 1. Age3p plays a major role in azole tolerance in C. albicans.

(A) Minimal Inhibitory Concentration (MIC) assay in rich YPD media showing that age3 cells are initially almost equally sensitive to FCZ compared to WT and revertant strains (24 hours reading, top), but fail to grow above this MIC threshold after prolonged incubation (72 hours reading, middle). MIC assays with two-fold serially diluted drug concentrations were done in duplicate and optical densities were normalized to drug-free control wells (see color bar). After 72 hours of incubation, 2 µl of each well of the MIC assay was spotted on fresh YPD media to assess the extent to which cells recover from the drug treatments (bottom). YPD recovery plates were incubated for 24 hours at 30°C. (B) FCZ sensitivity assayed on solid YPD media. No age3 colonies grew on YPD plates containing 24 hours-supra-MIC concentrations of FCZ. Overnight cultures were adjusted to OD₆₀₀ of 0.1, and then serially diluted four-fold, before 2 µl were spotted on plates. Plates were incubated for 48 hours at 30°C. (C) Time-kill curves in YPD media confirming that knocking out AGE3 abrogates tolerance to FCZ. The number of viable age3 cells decreases slightly over time, while growth of WT cells in the presence of FCZ still occurs. FCZ was used at 10 µg/ml. Shown is the average of two independent experiments plus SD values. Note that age3 cells grow as efficiently as WT cells in the absence of drugs. (D) MIC assays in YPD media shows that age3 mutants are extremely sensitive to numerous azoles after 48 hours and mildly more sensitive to non-azole ergosterol inhibitors (terbinafine, amphotericin B) as well as cell wall inhibitors when compared to WT cells. Fold reduction represents the ratio of the MIC value for WT over the MIC value of the age3 mutant. (E) age3 cells show differential sensitivity to different cell wall inhibitors on YPD media plates. The assay was done as described in (B).

Figure 2. Deleting AGE3 overrides clinical drug resistance and the calcineurin pathway.

(A) When AGE3 is knocked out in drug resistant clinical isolates F5 and S2, FCZ sensitivity is restored even below WT levels on solid YPD media. The assay was performed and analyzed as described in Figure 1B except plates were photographed after 24 hours. GOF = gain of function. (B) Calcineurin signaling stimulated either by extracellular CaCl₂ or by a constitutively active mutation in strain DSY2146 leads to FCZ resistance. age3 mutants do not respond to extracellular CaCl₂, while knocking out AGE3 in strain DSY2146 restored FCZ sensitivity. Disc diffusion assays were done by plating 2×10⁵ cells on YPD plates followed by applying discs containing 50 mg of FCZ to the surface of agar. Plates were incubated for 24 hours at 30°C.

Figure 3. Pharmacological inhibition of ARF cycling results in a potent, fungicidal synergy in combination with FCZ in C. albicans.

(A) Time-kill curves demonstrating that, while combining FCZ and BFA was initially equally efficacious in pathogen killing compared to combining FCZ and calcineurin inhibitors (FK506 or CsA), extended drug exposure only remained efficacious in pairwise BFA/FCZ combinations. Triple drug combinations of BFA/FCZ/calcineurin inhibitors were only initially (24 hours) more efficacious, but at 72 hours appeared equally efficacious compared to BFA/FCZ. The assay was done in YPD media. Drugs were used at 10 µg/ml for FCZ, 15 µg/ml for BFA, 1 µg/ml for CsA and 1 µg/ml for FK506. (B) Dose-matrix titration assay confirming the FCZ/BFA synergy in WT and drug resistant clinical isolates 5674, S2 and F5 in rich YPD media (top). Dose-matrix titration plates were incubated for 72 hours after which aliquots of each well were spotted on fresh YPD recovery plates (bottom). No-growth of recovery plates confirmed fungal cell death of the drug synergy. Recovery plates were incubated for 24 hours. Dose-matrix titration assays were analyzed as described for MIC assays in Figure 1A.

Figure 4. Pharmacological compromise of ARF cycling synergizes with various azoles as well as the cell wall inhibitor CF.

(A) MIC assays in YPD media demonstrated that WT C. albicans shows tolerance to MICO and KETO (top, compare 24 hours to 72 hours MIC readings). Combining BFA with either MICO or KETO resulted in a similarly potent fungicidal combination compared to BFA/FCZ (bottom). (B) CF showed trailing growth in rich YPD media (top) and synergized with BFA in a dose-matrix titration assay (bottom) against WT C. albicans. MIC and dose-matrix titration assays were performed and analyzed as described in Figure 1A and 3B, respectively.

Figure 5. BFA synergizes with different azoles in pathogenic non-albicans Candida species.

(A) When treated with FCZ, MICO or KETO, C. tropicalis, C. parapsilosis and C. glabrata isolates showed prominent growth above the initial MIC reading after extended incubation (24 hours vs. 72 hours). Dose-matrix titration assays confirming that BFA synergized with the three azoles in all three species. (B) No obvious tolerance effect was observed in C. krusei to any azoles tested and no synergy was observed when BFA was combined with these azoles. MIC and dose-matrix titration assays were performed and analyzed as described in Figure 1A and 3B.

Figure 6. BFA synergism with the fungistatic cell wall inhibitor CF in A. fumigatus.

BFA/CF interaction in an A. fumigatus disk diffusion assay on half-strength YPD media. CF alone creates an inhibition zone that still allows fungal growth. Combining CF and BFA abrogated growth within that zone. Discs containing 160 µg CF were applied after 10⁵ conidia were plated on plates containing water only, vehicle control (DMSO) or BFA (16 µg/ml), as indicated. Plates were incubated for 48 hours.

Figure 7. Genetic compromise of ARF cycling in C. albicans results in avirulence in a WT mouse model of disseminated disease.

(A) C. albicans WT-infected mice become gradually moribund up to day 11, while mice infected with age3 mutants did not show any clinical signs until the end of the experiment on day 21. The dotted blue line indicates that half of the age3 mutant-infected mice were sacrificed to compare fungal load. Those mice were not moribund. (B) On average, fungal load of WT-infected mice, when moribund, is significantly higher compared to mutant fungal burden taken at indicated times. (C) Kidney section of WT-infected mice (left) showing fungal hyphal formation, which is also seen in mutant-infected kidneys (right). Kidneys were collected on day 11 for histological examination. Ten mice were used per experimental group and monitored according to approved standards.

Figure 8. age3 mutants are attenuated in virulence in A/J mice and FCZ treatment significantly extends survival of age3-infected A/J mice.

(A) age3 mutant-infected mice survive significantly longer with a median survival of two days versus one day for WT and revertant-infected mice. Six mice were used per experimental group. (B) Fungal kidney burden was examined from moribund mice and was significantly higher in age3 mutant recovered cells compared to WT or revertant control groups. (C) A short FCZ therapy (4.5 mg/kg intraperitoneally once immediately after fungal infection, once on day one and once on day two post fungal infection) is significantly more efficacious when ARF cycling is genetically compromised as only the majority (83%) of mutant-infected mice survive until the end of the experiment (day 21). Six mice were used for WT and revertant groups and 12 mice for the age3 mutant group.