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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

Stt4 genetic interactions with Hsp90 are mediated through Wal1 regulation of actin.(A) Stt4 is not a client protein of Hsp90. Protein levels of N-terminally FLAG-tagged Stt4 were monitored in the tetO-HSP90/hsp90Δ background. Strains were incubated with or without 0.5 μg/mL DOX for 24 hours to deplete Hsp90 before protein extraction. Hsp90 levels are detected using an antibody against C. albicans Hsp90. Representative blots are shown here, with tubulin as a loading control. (B) Ypp1 and Efr3 are two additional members of the Stt4 complex that influence tolerance to geldanamycin. MIC assays were performed as described in Fig 4. (C) The PH-domain proteins Osh3 and Wal1 are genetic interactors with Hsp90. MIC assays were performed as described in Fig 4. (D) Stt4 and Wal1 are required for normal actin localization in the cell. Strains were sub-cultured in RPMI at 37°C for 6 hours before fixation and staining with rhodamine-phalloidin for F-actin. (E) Actin inhibitors are synergistic with Hsp90 inhibitors. Dose response matrixes were performed in RPMI at 37°C and incubated for 38 hours.
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pgen.1006142.g005: Stt4 genetic interactions with Hsp90 are mediated through Wal1 regulation of actin.(A) Stt4 is not a client protein of Hsp90. Protein levels of N-terminally FLAG-tagged Stt4 were monitored in the tetO-HSP90/hsp90Δ background. Strains were incubated with or without 0.5 μg/mL DOX for 24 hours to deplete Hsp90 before protein extraction. Hsp90 levels are detected using an antibody against C. albicans Hsp90. Representative blots are shown here, with tubulin as a loading control. (B) Ypp1 and Efr3 are two additional members of the Stt4 complex that influence tolerance to geldanamycin. MIC assays were performed as described in Fig 4. (C) The PH-domain proteins Osh3 and Wal1 are genetic interactors with Hsp90. MIC assays were performed as described in Fig 4. (D) Stt4 and Wal1 are required for normal actin localization in the cell. Strains were sub-cultured in RPMI at 37°C for 6 hours before fixation and staining with rhodamine-phalloidin for F-actin. (E) Actin inhibitors are synergistic with Hsp90 inhibitors. Dose response matrixes were performed in RPMI at 37°C and incubated for 38 hours.

Mentions: C. albicans Stt4 is a type III phosphatidylinositol-4-kinase (PI4K); previous work in mammalian cells demonstrated that the type IIβ PI4K protein is stabilized by Hsp90 [34]. Therefore, we examined the stability of CaStt4 upon Hsp90 depletion. To do so, we engineered an N-terminally FLAG-tagged STT4 allele under the ACT1 promoter in the tetO-HSP90/hsp90Δ background, and monitored FLAG-Stt4 protein levels upon transcriptional repression of HSP90 with doxycycline (Fig 5A). There was no reduction in FLAG-Stt4 levels upon Hsp90 depletion, indicating that that unlike in mammalian cells, Stt4 is not a client protein of Hsp90 in C. albicans.


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)

Stt4 genetic interactions with Hsp90 are mediated through Wal1 regulation of actin.(A) Stt4 is not a client protein of Hsp90. Protein levels of N-terminally FLAG-tagged Stt4 were monitored in the tetO-HSP90/hsp90Δ background. Strains were incubated with or without 0.5 μg/mL DOX for 24 hours to deplete Hsp90 before protein extraction. Hsp90 levels are detected using an antibody against C. albicans Hsp90. Representative blots are shown here, with tubulin as a loading control. (B) Ypp1 and Efr3 are two additional members of the Stt4 complex that influence tolerance to geldanamycin. MIC assays were performed as described in Fig 4. (C) The PH-domain proteins Osh3 and Wal1 are genetic interactors with Hsp90. MIC assays were performed as described in Fig 4. (D) Stt4 and Wal1 are required for normal actin localization in the cell. Strains were sub-cultured in RPMI at 37°C for 6 hours before fixation and staining with rhodamine-phalloidin for F-actin. (E) Actin inhibitors are synergistic with Hsp90 inhibitors. Dose response matrixes were performed in RPMI at 37°C and incubated for 38 hours.
© Copyright Policy
Related In: Results  -  Collection

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

pgen.1006142.g005: Stt4 genetic interactions with Hsp90 are mediated through Wal1 regulation of actin.(A) Stt4 is not a client protein of Hsp90. Protein levels of N-terminally FLAG-tagged Stt4 were monitored in the tetO-HSP90/hsp90Δ background. Strains were incubated with or without 0.5 μg/mL DOX for 24 hours to deplete Hsp90 before protein extraction. Hsp90 levels are detected using an antibody against C. albicans Hsp90. Representative blots are shown here, with tubulin as a loading control. (B) Ypp1 and Efr3 are two additional members of the Stt4 complex that influence tolerance to geldanamycin. MIC assays were performed as described in Fig 4. (C) The PH-domain proteins Osh3 and Wal1 are genetic interactors with Hsp90. MIC assays were performed as described in Fig 4. (D) Stt4 and Wal1 are required for normal actin localization in the cell. Strains were sub-cultured in RPMI at 37°C for 6 hours before fixation and staining with rhodamine-phalloidin for F-actin. (E) Actin inhibitors are synergistic with Hsp90 inhibitors. Dose response matrixes were performed in RPMI at 37°C and incubated for 38 hours.
Mentions: C. albicans Stt4 is a type III phosphatidylinositol-4-kinase (PI4K); previous work in mammalian cells demonstrated that the type IIβ PI4K protein is stabilized by Hsp90 [34]. Therefore, we examined the stability of CaStt4 upon Hsp90 depletion. To do so, we engineered an N-terminally FLAG-tagged STT4 allele under the ACT1 promoter in the tetO-HSP90/hsp90Δ background, and monitored FLAG-Stt4 protein levels upon transcriptional repression of HSP90 with doxycycline (Fig 5A). There was no reduction in FLAG-Stt4 levels upon Hsp90 depletion, indicating that that unlike in mammalian cells, Stt4 is not a client protein of Hsp90 in C. albicans.

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