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.
Highlights
Invasive fungal infections pose a serious health risk to hospitalized patients worldwide
Fungal infections are often treated with azole drugs, but due to the fungistatic nature of these agents, C. albicans can develop drug resistance, leading to therapy failure
To improve the action of azoles and convert them into fungicidal drugs, we first systematically analyzed the genetic requirements for tolerance to one such azole drug, fluconazole. Both genetically and pharmacologically, that components of the ARF cycling machinery are critical in mediating both azole and echinocandin tolerance in C. albicans as well as several other pathogenic Candida species and in the pathogenic mold Aspergillus fumigatus
Summary
Invasive fungal infections pose a serious health risk to hospitalized patients worldwide. Three classes of antifungal drugs are suitable for treatment of systemic infections caused by these fungi: polyenes (most notably amphotericin B) and azoles (e.g. fluconazole, FCZ) have been applied for decades, while the echinocandins (e.g. caspofungin, CF) represent a new class of antifungal that has entered treatment regimes over the past 10 years [10,11] While these therapy options can be effective, they exhibit several shortcomings. The major sterol in fungal cell membranes, is analogous to the mammalian lipid cholesterol, this strategy, when amphotericin B is applied, can be problematic due to host toxicity [10] Another complication of current antifungal strategies is that available drugs each possess a different spectrum of antifungal activities. Most importantly, the small number of treatment options available has resulted in widespread drug
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