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

Elias Epp, Ghyslaine Vanier, Doreen Harcus, Anna Y Lee, Gregor Jansen, Michael Hallett, Don C Sheppard, David Y Thomas, Carol A Munro, Alaka Mullick, Malcolm Whiteway

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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.
Original languageEnglish
Article numbere1000753
Number of pages15
JournalPLoS Pathogens
Volume6
Issue number2
DOIs
Publication statusPublished - 5 Feb 2010

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Essential Drugs
Reverse Genetics
Fluconazole
Candida albicans
Drug Resistance
Virulence
Brefeldin A
ADP-Ribosylation Factors
Pharmaceutical Preparations
caspofungin
Fungal Drug Resistance
GTPase-Activating Proteins
Guanine Nucleotide Exchange Factors
Aspergillus fumigatus
Calcineurin
Sterols
Therapeutics
Candida
Cell Wall
Saccharomyces cerevisiae

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Epp, E., Vanier, G., Harcus, D., Lee, A. Y., Jansen, G., Hallett, M., ... Whiteway, M. (2010). Reverse genetics in Candida albicans predicts ARF cycling is essential for drug resistance and virulence. PLoS Pathogens, 6(2), [e1000753]. https://doi.org/10.1371/journal.ppat.1000753

Reverse genetics in Candida albicans predicts ARF cycling is essential for drug resistance and virulence. / Epp, Elias; Vanier, Ghyslaine; Harcus, Doreen; Lee, Anna Y; Jansen, Gregor; Hallett, Michael; Sheppard, Don C; Thomas, David Y; Munro, Carol A; Mullick, Alaka; Whiteway, Malcolm.

In: PLoS Pathogens, Vol. 6, No. 2, e1000753, 05.02.2010.

Research output: Contribution to journalArticle

Epp, E, Vanier, G, Harcus, D, Lee, AY, Jansen, G, Hallett, M, Sheppard, DC, Thomas, DY, Munro, CA, Mullick, A & Whiteway, M 2010, 'Reverse genetics in Candida albicans predicts ARF cycling is essential for drug resistance and virulence', PLoS Pathogens, vol. 6, no. 2, e1000753. https://doi.org/10.1371/journal.ppat.1000753
Epp, Elias ; Vanier, Ghyslaine ; Harcus, Doreen ; Lee, Anna Y ; Jansen, Gregor ; Hallett, Michael ; Sheppard, Don C ; Thomas, David Y ; Munro, Carol A ; Mullick, Alaka ; Whiteway, Malcolm. / Reverse genetics in Candida albicans predicts ARF cycling is essential for drug resistance and virulence. In: PLoS Pathogens. 2010 ; Vol. 6, No. 2.
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