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Mapping the Hsp90 Genetic Network Reveals Ergosterol Biosynthesis and Phosphatidylinositol-4-Kinase Signaling as Core Circuitry Governing Cellular Stress.

O'Meara TR, Veri AO, Polvi EJ, Li X, Valaei SF, Diezmann S, Cowen LE - PLoS Genet. (2016)

Bottom Line: Consistent with this model, actin inhibitors are synergistic with Hsp90 inhibitors.We highlight new connections between Hsp90 and virulence traits, demonstrating that Erg5 and Stt4 enable activation of macrophage pyroptosis.This work uncovers novel circuitry regulating Hsp90 functional capacity and new effectors governing drug resistance, morphogenesis and virulence, revealing new targets for antifungal drug development.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
Candida albicans is a leading human fungal pathogen that causes life-threatening systemic infections. A key regulator of C. albicans stress response, drug resistance, morphogenesis, and virulence is the molecular chaperone Hsp90. Targeting Hsp90 provides a powerful strategy to treat fungal infections, however, the therapeutic utility of current inhibitors is compromised by toxicity due to inhibition of host Hsp90. To identify components of the Hsp90-dependent circuitry governing virulence and drug resistance that are sufficiently divergent for selective targeting in the pathogen, we pioneered chemical genomic profiling of the Hsp90 genetic network in C. albicans. Here, we screen mutant collections covering ~10% of the genome for hypersensitivity to Hsp90 inhibition in multiple environmental conditions. We identify 158 HSP90 chemical genetic interactors, most of which are important for growth only in specific environments. We discovered that the sterol C-22 desaturase gene ERG5 and the phosphatidylinositol-4-kinase (PI4K) gene STT4 are HSP90 genetic interactors under multiple conditions, suggesting a function upstream of Hsp90. By systematic analysis of the ergosterol biosynthetic cascade, we demonstrate that defects in ergosterol biosynthesis induce cellular stress that overwhelms Hsp90's functional capacity. By analysis of the phosphatidylinositol pathway, we demonstrate that there is a genetic interaction between the PI4K Stt4 and Hsp90. We also establish that Stt4 is required for normal actin polarization through regulation of Wal1, and suggest a model in which defects in actin remodeling induces stress that creates a cellular demand for Hsp90 that exceeds its functional capacity. Consistent with this model, actin inhibitors are synergistic with Hsp90 inhibitors. We highlight new connections between Hsp90 and virulence traits, demonstrating that Erg5 and Stt4 enable activation of macrophage pyroptosis. This work uncovers novel circuitry regulating Hsp90 functional capacity and new effectors governing drug resistance, morphogenesis and virulence, revealing new targets for antifungal drug development.

No MeSH data available.


Related in: MedlinePlus

Mapping the C. albicans Hsp90 genetic interaction network.(A) Chemical genomic screening revealed 11 mutants that are hypersensitive to Hsp90 inhibition. Strains were screened at 3 μM geldanamycin in RPMI at 37°C, and percent growth is normalized to the no drug condition. * indicates p <0.05 compared to the wild-type strain using t-tests. (B) The HSP90 genetic interaction network is environmentally contingent. The network is composed of 158 genetic interactors identified in five different growth conditions (grey boxes). Each HSP90 genetic interactor is indicated by a box, with edges connecting it to the environmental conditions in which it interacts with HSP90. The color of the box reflects the number of conditions in which the mutant demonstrates hypersensitivity to Hsp90 inhibition, with the majority of interactions only occurring under a single environmental condition. FLU = fluconazole, C = caspofungin, T = tunicamycin. Thick black outline = screened at 1 μM geldanamycin except for stt4Δ/Δ which was screened at 0.375 μM geldanamycin.
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pgen.1006142.g001: Mapping the C. albicans Hsp90 genetic interaction network.(A) Chemical genomic screening revealed 11 mutants that are hypersensitive to Hsp90 inhibition. Strains were screened at 3 μM geldanamycin in RPMI at 37°C, and percent growth is normalized to the no drug condition. * indicates p <0.05 compared to the wild-type strain using t-tests. (B) The HSP90 genetic interaction network is environmentally contingent. The network is composed of 158 genetic interactors identified in five different growth conditions (grey boxes). Each HSP90 genetic interactor is indicated by a box, with edges connecting it to the environmental conditions in which it interacts with HSP90. The color of the box reflects the number of conditions in which the mutant demonstrates hypersensitivity to Hsp90 inhibition, with the majority of interactions only occurring under a single environmental condition. FLU = fluconazole, C = caspofungin, T = tunicamycin. Thick black outline = screened at 1 μM geldanamycin except for stt4Δ/Δ which was screened at 0.375 μM geldanamycin.

Mentions: We extended our previous analysis of the HSP90 genetic interaction network in C. albicans, utilizing the same chemical genomic approach to screen two additional mutant libraries that cover 772 genes, including 566 ORFs distinct from the previous screen [11,21]. An advantage of these libraries over that used in our previous analysis is that these are composed of precise homozygous deletion mutants rather than transposon insertion mutants. Each strain was screened for growth under standard host-relevant conditions (RPMI, 37°C) in the presence and absence of a low concentration of the pharmacological Hsp90 inhibitor geldanamycin (3 μM) that does not affect growth of the wild-type strain. This revealed 11 genes that are strong genetic interactors of Hsp90 (Fig 1A), as the corresponding mutants had at least 50% reduction in growth in response to this low dose of Hsp90 inhibition. Of these, mutants lacking the sterol C-22 desaturase gene ERG5 and the phosphatidylinositol-4-kinase (PI4K) gene STT4 demonstrated the greatest sensitivity to Hsp90 inhibition. These genetic interactions were confirmed using Gene Replacement And Conditional Expression (GRACE) strains [22,23], whose target gene expression is repressible using the tetracycline analog doxycycline (S1 Fig). This confirms that the sensitivity of the stt4Δ/Δ and erg5Δ/Δ mutants to geldanamycin is due to the specific genetic perturbation and not to spurious mutations.


Mapping the Hsp90 Genetic Network Reveals Ergosterol Biosynthesis and Phosphatidylinositol-4-Kinase Signaling as Core Circuitry Governing Cellular Stress.

O'Meara TR, Veri AO, Polvi EJ, Li X, Valaei SF, Diezmann S, Cowen LE - PLoS Genet. (2016)

Mapping the C. albicans Hsp90 genetic interaction network.(A) Chemical genomic screening revealed 11 mutants that are hypersensitive to Hsp90 inhibition. Strains were screened at 3 μM geldanamycin in RPMI at 37°C, and percent growth is normalized to the no drug condition. * indicates p <0.05 compared to the wild-type strain using t-tests. (B) The HSP90 genetic interaction network is environmentally contingent. The network is composed of 158 genetic interactors identified in five different growth conditions (grey boxes). Each HSP90 genetic interactor is indicated by a box, with edges connecting it to the environmental conditions in which it interacts with HSP90. The color of the box reflects the number of conditions in which the mutant demonstrates hypersensitivity to Hsp90 inhibition, with the majority of interactions only occurring under a single environmental condition. FLU = fluconazole, C = caspofungin, T = tunicamycin. Thick black outline = screened at 1 μM geldanamycin except for stt4Δ/Δ which was screened at 0.375 μM geldanamycin.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4920384&req=5

pgen.1006142.g001: Mapping the C. albicans Hsp90 genetic interaction network.(A) Chemical genomic screening revealed 11 mutants that are hypersensitive to Hsp90 inhibition. Strains were screened at 3 μM geldanamycin in RPMI at 37°C, and percent growth is normalized to the no drug condition. * indicates p <0.05 compared to the wild-type strain using t-tests. (B) The HSP90 genetic interaction network is environmentally contingent. The network is composed of 158 genetic interactors identified in five different growth conditions (grey boxes). Each HSP90 genetic interactor is indicated by a box, with edges connecting it to the environmental conditions in which it interacts with HSP90. The color of the box reflects the number of conditions in which the mutant demonstrates hypersensitivity to Hsp90 inhibition, with the majority of interactions only occurring under a single environmental condition. FLU = fluconazole, C = caspofungin, T = tunicamycin. Thick black outline = screened at 1 μM geldanamycin except for stt4Δ/Δ which was screened at 0.375 μM geldanamycin.
Mentions: We extended our previous analysis of the HSP90 genetic interaction network in C. albicans, utilizing the same chemical genomic approach to screen two additional mutant libraries that cover 772 genes, including 566 ORFs distinct from the previous screen [11,21]. An advantage of these libraries over that used in our previous analysis is that these are composed of precise homozygous deletion mutants rather than transposon insertion mutants. Each strain was screened for growth under standard host-relevant conditions (RPMI, 37°C) in the presence and absence of a low concentration of the pharmacological Hsp90 inhibitor geldanamycin (3 μM) that does not affect growth of the wild-type strain. This revealed 11 genes that are strong genetic interactors of Hsp90 (Fig 1A), as the corresponding mutants had at least 50% reduction in growth in response to this low dose of Hsp90 inhibition. Of these, mutants lacking the sterol C-22 desaturase gene ERG5 and the phosphatidylinositol-4-kinase (PI4K) gene STT4 demonstrated the greatest sensitivity to Hsp90 inhibition. These genetic interactions were confirmed using Gene Replacement And Conditional Expression (GRACE) strains [22,23], whose target gene expression is repressible using the tetracycline analog doxycycline (S1 Fig). This confirms that the sensitivity of the stt4Δ/Δ and erg5Δ/Δ mutants to geldanamycin is due to the specific genetic perturbation and not to spurious mutations.

Bottom Line: Consistent with this model, actin inhibitors are synergistic with Hsp90 inhibitors.We highlight new connections between Hsp90 and virulence traits, demonstrating that Erg5 and Stt4 enable activation of macrophage pyroptosis.This work uncovers novel circuitry regulating Hsp90 functional capacity and new effectors governing drug resistance, morphogenesis and virulence, revealing new targets for antifungal drug development.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada.

ABSTRACT
Candida albicans is a leading human fungal pathogen that causes life-threatening systemic infections. A key regulator of C. albicans stress response, drug resistance, morphogenesis, and virulence is the molecular chaperone Hsp90. Targeting Hsp90 provides a powerful strategy to treat fungal infections, however, the therapeutic utility of current inhibitors is compromised by toxicity due to inhibition of host Hsp90. To identify components of the Hsp90-dependent circuitry governing virulence and drug resistance that are sufficiently divergent for selective targeting in the pathogen, we pioneered chemical genomic profiling of the Hsp90 genetic network in C. albicans. Here, we screen mutant collections covering ~10% of the genome for hypersensitivity to Hsp90 inhibition in multiple environmental conditions. We identify 158 HSP90 chemical genetic interactors, most of which are important for growth only in specific environments. We discovered that the sterol C-22 desaturase gene ERG5 and the phosphatidylinositol-4-kinase (PI4K) gene STT4 are HSP90 genetic interactors under multiple conditions, suggesting a function upstream of Hsp90. By systematic analysis of the ergosterol biosynthetic cascade, we demonstrate that defects in ergosterol biosynthesis induce cellular stress that overwhelms Hsp90's functional capacity. By analysis of the phosphatidylinositol pathway, we demonstrate that there is a genetic interaction between the PI4K Stt4 and Hsp90. We also establish that Stt4 is required for normal actin polarization through regulation of Wal1, and suggest a model in which defects in actin remodeling induces stress that creates a cellular demand for Hsp90 that exceeds its functional capacity. Consistent with this model, actin inhibitors are synergistic with Hsp90 inhibitors. We highlight new connections between Hsp90 and virulence traits, demonstrating that Erg5 and Stt4 enable activation of macrophage pyroptosis. This work uncovers novel circuitry regulating Hsp90 functional capacity and new effectors governing drug resistance, morphogenesis and virulence, revealing new targets for antifungal drug development.

No MeSH data available.


Related in: MedlinePlus