Reverse genetics in Candida albicans predicts ARF cycling is essential for drug resistance and virulence.
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 combine...
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Public Library of Science (PLoS)
2010-02-01
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| Series: | PLoS Pathogens |
| Online Access: | https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1000753&type=printable |
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| author | 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 |
| author_facet | 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 |
| author_sort | Elias Epp |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-e0fc33be978240cdb11ed15d4e4d4ff3 |
| institution | OA Journals |
| issn | 1553-7366 1553-7374 |
| language | English |
| publishDate | 2010-02-01 |
| publisher | Public Library of Science (PLoS) |
| record_format | Article |
| series | PLoS Pathogens |
| spelling | doaj-art-e0fc33be978240cdb11ed15d4e4d4ff32025-08-20T02:02:01ZengPublic Library of Science (PLoS)PLoS Pathogens1553-73661553-73742010-02-0162e100075310.1371/journal.ppat.1000753Reverse genetics in Candida albicans predicts ARF cycling is essential for drug resistance and virulence.Elias EppGhyslaine VanierDoreen HarcusAnna Y LeeGregor JansenMichael HallettDon C SheppardDavid Y ThomasCarol A MunroAlaka MullickMalcolm WhitewayCandida 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.https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1000753&type=printable |
| spellingShingle | 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 Reverse genetics in Candida albicans predicts ARF cycling is essential for drug resistance and virulence. PLoS Pathogens |
| title | Reverse genetics in Candida albicans predicts ARF cycling is essential for drug resistance and virulence. |
| title_full | Reverse genetics in Candida albicans predicts ARF cycling is essential for drug resistance and virulence. |
| title_fullStr | Reverse genetics in Candida albicans predicts ARF cycling is essential for drug resistance and virulence. |
| title_full_unstemmed | Reverse genetics in Candida albicans predicts ARF cycling is essential for drug resistance and virulence. |
| title_short | Reverse genetics in Candida albicans predicts ARF cycling is essential for drug resistance and virulence. |
| title_sort | reverse genetics in candida albicans predicts arf cycling is essential for drug resistance and virulence |
| url | https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1000753&type=printable |
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