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Genome-wide fitness test and mechanism-of-action studies of inhibitory compounds in Candida albicans.

Xu D, Jiang B, Ketela T, Lemieux S, Veillette K, Martel N, Davison J, Sillaots S, Trosok S, Bachewich C, Bussey H, Youngman P, Roemer T - PLoS Pathog. (2007)

Bottom Line: Candida albicans is a prevalent fungal pathogen amongst the immunocompromised population, causing both superficial and life-threatening infections.Since C. albicans is diploid, classical transmission genetics can not be performed to study specific aspects of its biology and pathogenesis.In this report, chemical-genetic relationships are provided for multiple FDA-approved antifungal drugs (fluconazole, voriconazole, caspofungin, 5-fluorocytosine, and amphotericin B) as well as additional compounds targeting ergosterol, fatty acid and sphingolipid biosynthesis, microtubules, actin, secretion, rRNA processing, translation, glycosylation, and protein folding mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Center of Fungal Genetics, Merck Frosst Canada Ltd., Montreal, Quebec, Canada.

ABSTRACT
Candida albicans is a prevalent fungal pathogen amongst the immunocompromised population, causing both superficial and life-threatening infections. Since C. albicans is diploid, classical transmission genetics can not be performed to study specific aspects of its biology and pathogenesis. Here, we exploit the diploid status of C. albicans by constructing a library of 2,868 heterozygous deletion mutants and screening this collection using 35 known or novel compounds to survey chemically induced haploinsufficiency in the pathogen. In this reverse genetic assay termed the fitness test, genes related to the mechanism of action of the probe compounds are clearly identified, supporting their functional roles and genetic interactions. In this report, chemical-genetic relationships are provided for multiple FDA-approved antifungal drugs (fluconazole, voriconazole, caspofungin, 5-fluorocytosine, and amphotericin B) as well as additional compounds targeting ergosterol, fatty acid and sphingolipid biosynthesis, microtubules, actin, secretion, rRNA processing, translation, glycosylation, and protein folding mechanisms. We also demonstrate how chemically induced haploinsufficiency profiles can be used to identify the mechanism of action of novel antifungal agents, thereby illustrating the potential utility of this approach to antifungal drug discovery.

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Related in: MedlinePlus

CaFT Profiling and Characterization of Cerulenin-Induced HI(A) CaFT profiles of cerulenin, with highlighted strains as follows: FAS1 (orf19.979), FAS2 (orf19.5949), and MDR1 (orf19.5604). A homozygous deletion of MDR1 is hypersensitive to cerulenin [21]; however, the heterozygous deletion strain showed no specific hypersensitivity at the ICs tested in the CaFT.(B) The spot tests of selected heterozygous deletion strains against cerulenin. Note that the hypersensitivity of the MDR1 strain was only seen at the highest concentration of cerulenin tested.(C) A model for regulating stoichiometry of the FAS complex in S. cerevisiae. As shown by Wenz et al. [20], the expression of ScFAS2 is repressed by an unknown transcription repressor (rep. X), which is in turn derepressed by free β subunit (Fas1p). According to this model, ScFAS2 expression is dependent on free Fas1p to control the normal stoichiometry of the FAS complex. A similar regulatory mechanism in C. albicans may explain the observed cerulenin-induced HI of FAS1 but not FAS2 (see text for details).(D) FAS1 is haploinsufficient under the standard growth conditions. In order to determine HI, cultures of the selected strains were first incubated for 6 h (reaching exponential growth) and then diluted to an OD600 of 0.005. The fresh cultures were incubated for another 12 h, after which the OD was monitored for an additional 4 h.
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ppat-0030092-g003: CaFT Profiling and Characterization of Cerulenin-Induced HI(A) CaFT profiles of cerulenin, with highlighted strains as follows: FAS1 (orf19.979), FAS2 (orf19.5949), and MDR1 (orf19.5604). A homozygous deletion of MDR1 is hypersensitive to cerulenin [21]; however, the heterozygous deletion strain showed no specific hypersensitivity at the ICs tested in the CaFT.(B) The spot tests of selected heterozygous deletion strains against cerulenin. Note that the hypersensitivity of the MDR1 strain was only seen at the highest concentration of cerulenin tested.(C) A model for regulating stoichiometry of the FAS complex in S. cerevisiae. As shown by Wenz et al. [20], the expression of ScFAS2 is repressed by an unknown transcription repressor (rep. X), which is in turn derepressed by free β subunit (Fas1p). According to this model, ScFAS2 expression is dependent on free Fas1p to control the normal stoichiometry of the FAS complex. A similar regulatory mechanism in C. albicans may explain the observed cerulenin-induced HI of FAS1 but not FAS2 (see text for details).(D) FAS1 is haploinsufficient under the standard growth conditions. In order to determine HI, cultures of the selected strains were first incubated for 6 h (reaching exponential growth) and then diluted to an OD600 of 0.005. The fresh cultures were incubated for another 12 h, after which the OD was monitored for an additional 4 h.

Mentions: Cerulenin specifically inhibits the condensation reaction associated with the α subunit of the fatty acid synthase (FAS), a heteromultimeric complex of α (Fas2p) and β (Fas1p) subunits. Although not examined in the ScFT, cerulenin elicited reproducibly specific hypersensitivity of the FAS1 strain but not of FAS2, even at the highest drug concentration tested (Figure 3A). These results were also demonstrated by spot tests (Figure 3B). In S. cerevisiae, the expression of ScFAS2 is regulated in an ScFas1p-dependent manner to control the stoichiometry of the FAS complex [20]. Our results suggest that a similar regulatory mechanism exists in C. albicans, with the level of Fas1p being the critical factor controlling the FAS complex (see Figure 3C and Discussion). Consistent with this model, only FAS1 exhibits HI under the standard growth conditions (Figure 3B and 3D).


Genome-wide fitness test and mechanism-of-action studies of inhibitory compounds in Candida albicans.

Xu D, Jiang B, Ketela T, Lemieux S, Veillette K, Martel N, Davison J, Sillaots S, Trosok S, Bachewich C, Bussey H, Youngman P, Roemer T - PLoS Pathog. (2007)

CaFT Profiling and Characterization of Cerulenin-Induced HI(A) CaFT profiles of cerulenin, with highlighted strains as follows: FAS1 (orf19.979), FAS2 (orf19.5949), and MDR1 (orf19.5604). A homozygous deletion of MDR1 is hypersensitive to cerulenin [21]; however, the heterozygous deletion strain showed no specific hypersensitivity at the ICs tested in the CaFT.(B) The spot tests of selected heterozygous deletion strains against cerulenin. Note that the hypersensitivity of the MDR1 strain was only seen at the highest concentration of cerulenin tested.(C) A model for regulating stoichiometry of the FAS complex in S. cerevisiae. As shown by Wenz et al. [20], the expression of ScFAS2 is repressed by an unknown transcription repressor (rep. X), which is in turn derepressed by free β subunit (Fas1p). According to this model, ScFAS2 expression is dependent on free Fas1p to control the normal stoichiometry of the FAS complex. A similar regulatory mechanism in C. albicans may explain the observed cerulenin-induced HI of FAS1 but not FAS2 (see text for details).(D) FAS1 is haploinsufficient under the standard growth conditions. In order to determine HI, cultures of the selected strains were first incubated for 6 h (reaching exponential growth) and then diluted to an OD600 of 0.005. The fresh cultures were incubated for another 12 h, after which the OD was monitored for an additional 4 h.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1904411&req=5

ppat-0030092-g003: CaFT Profiling and Characterization of Cerulenin-Induced HI(A) CaFT profiles of cerulenin, with highlighted strains as follows: FAS1 (orf19.979), FAS2 (orf19.5949), and MDR1 (orf19.5604). A homozygous deletion of MDR1 is hypersensitive to cerulenin [21]; however, the heterozygous deletion strain showed no specific hypersensitivity at the ICs tested in the CaFT.(B) The spot tests of selected heterozygous deletion strains against cerulenin. Note that the hypersensitivity of the MDR1 strain was only seen at the highest concentration of cerulenin tested.(C) A model for regulating stoichiometry of the FAS complex in S. cerevisiae. As shown by Wenz et al. [20], the expression of ScFAS2 is repressed by an unknown transcription repressor (rep. X), which is in turn derepressed by free β subunit (Fas1p). According to this model, ScFAS2 expression is dependent on free Fas1p to control the normal stoichiometry of the FAS complex. A similar regulatory mechanism in C. albicans may explain the observed cerulenin-induced HI of FAS1 but not FAS2 (see text for details).(D) FAS1 is haploinsufficient under the standard growth conditions. In order to determine HI, cultures of the selected strains were first incubated for 6 h (reaching exponential growth) and then diluted to an OD600 of 0.005. The fresh cultures were incubated for another 12 h, after which the OD was monitored for an additional 4 h.
Mentions: Cerulenin specifically inhibits the condensation reaction associated with the α subunit of the fatty acid synthase (FAS), a heteromultimeric complex of α (Fas2p) and β (Fas1p) subunits. Although not examined in the ScFT, cerulenin elicited reproducibly specific hypersensitivity of the FAS1 strain but not of FAS2, even at the highest drug concentration tested (Figure 3A). These results were also demonstrated by spot tests (Figure 3B). In S. cerevisiae, the expression of ScFAS2 is regulated in an ScFas1p-dependent manner to control the stoichiometry of the FAS complex [20]. Our results suggest that a similar regulatory mechanism exists in C. albicans, with the level of Fas1p being the critical factor controlling the FAS complex (see Figure 3C and Discussion). Consistent with this model, only FAS1 exhibits HI under the standard growth conditions (Figure 3B and 3D).

Bottom Line: Candida albicans is a prevalent fungal pathogen amongst the immunocompromised population, causing both superficial and life-threatening infections.Since C. albicans is diploid, classical transmission genetics can not be performed to study specific aspects of its biology and pathogenesis.In this report, chemical-genetic relationships are provided for multiple FDA-approved antifungal drugs (fluconazole, voriconazole, caspofungin, 5-fluorocytosine, and amphotericin B) as well as additional compounds targeting ergosterol, fatty acid and sphingolipid biosynthesis, microtubules, actin, secretion, rRNA processing, translation, glycosylation, and protein folding mechanisms.

View Article: PubMed Central - PubMed

Affiliation: Center of Fungal Genetics, Merck Frosst Canada Ltd., Montreal, Quebec, Canada.

ABSTRACT
Candida albicans is a prevalent fungal pathogen amongst the immunocompromised population, causing both superficial and life-threatening infections. Since C. albicans is diploid, classical transmission genetics can not be performed to study specific aspects of its biology and pathogenesis. Here, we exploit the diploid status of C. albicans by constructing a library of 2,868 heterozygous deletion mutants and screening this collection using 35 known or novel compounds to survey chemically induced haploinsufficiency in the pathogen. In this reverse genetic assay termed the fitness test, genes related to the mechanism of action of the probe compounds are clearly identified, supporting their functional roles and genetic interactions. In this report, chemical-genetic relationships are provided for multiple FDA-approved antifungal drugs (fluconazole, voriconazole, caspofungin, 5-fluorocytosine, and amphotericin B) as well as additional compounds targeting ergosterol, fatty acid and sphingolipid biosynthesis, microtubules, actin, secretion, rRNA processing, translation, glycosylation, and protein folding mechanisms. We also demonstrate how chemically induced haploinsufficiency profiles can be used to identify the mechanism of action of novel antifungal agents, thereby illustrating the potential utility of this approach to antifungal drug discovery.

Show MeSH
Related in: MedlinePlus