Antimicrobial-induced DNA damage in bacterial and fungal pathogens.

<p>Sublethal doses of antimicrobial agents can directly or indirectly damage DNA in bacteria and fungi. In bacteria, DNA damage may lead to up-regulation of an SOS response, error-prone translesion DNA synthesis, or other stress responses that result in mutations including single nucleotide polymorphisms (SNPs) and the movement of mobile genetic elements. In fungi, treatment with antifungals can lead to DNA damage, resulting in homologous recombination and loss of heterozygosity (LOH), or other cellular stress responses, leading to unequal chromosomal segregation during mitosis and aneuploidy. Bacteria and fungi are not to scale.</p

The high-connectivity interactor Ahr1 influences <i>HSP90</i> expression and morphogenesis.

<p>(A) <i>HSP90</i> transcript levels are reduced in the <i>ahr1</i>Δ/<i>ahr1</i>Δ mutant. <i>HSP90</i> transcript levels were measured in the wild type (SN152), the <i>ahr1</i>Δ/<i>ahr1</i>Δ mutant, and the <i>AHR1</i> complemented strain by quantitative RT-PCR and normalized to <i>GPD1</i>. Data are means ± standard deviation for triplicate samples. (B) The <i>ahr1</i>Δ/<i>ahr1</i>Δ mutant filaments in rich medium at 30°C, consistent with the effects of compromised Hsp90 function. Differential Interference Contrast microscopy of strains incubated in rich medium at 30°C for 24 hours with or without 10 µM geldanamycin (GdA). Scale bar is 10 µm.</p

The protein kinase CK2 regulatory subunits regulate function of the Hsp90/Cdc37 protein complex.

<p>(A) Hsp90 serine and threonine phosphorylation is severely reduced in the <i>ckb1</i>Δ/<i>ckb1</i>Δ mutant, and Cdc37 serine and threonine phosphorylation is severely reduced in both the <i>ckb1</i>Δ/<i>ckb1</i>Δ and <i>ckb2</i>Δ/<i>ckb2</i>Δ mutants. Hsp90 or Cdc37 were immunoprecipitated and Western blots were hybridized with CaHsp90, TAP (to detect Cdc37-TAP), phosphothreonine, or phosphoserine antibodies. The ratio of phosphorylated to unphosphorylated Hsp90 or Cdc37 in each CK2 mutant was quantified relative to the wild type. (B) Western analysis demonstrates that Cdc37 levels are severely reduced (>50%, red box) in the mutant lacking the regulatory subunit Ckb2 (<i>ckb2</i>Δ/<i>ckb2</i>Δ) in the absence of external stress compared to the wild type (WT, BWP17); Hsp90 and Hog1 levels, however, are reduced (>25%, yellow box) in strains that lack the regulatory subunits (<i>ckb1</i>Δ/<i>ckb1</i>Δ or <i>ckb2</i>Δ/<i>ckb2</i>Δ) in response to oxidative stress in the form of a 10 minute treatment with 1 mM hydrogen peroxide. Actin served as loading control. (C) Deletion of CK2 regulatory subunits, <i>CKB1</i> or <i>CKB2</i>, phenocopies deletion of <i>HOG1</i> in terms of hypersensitivity to high osmolarity stress exerted by sorbitol. Growth is quantitatively displayed with color as indicated with the color bar. (D) Our results support a model in which the regulatory subunits of CK2 (Ckb1 and Ckb2) affect phosphorylation of Hsp90 and Cdc37, protein levels of the Hsp90-Cdc37 complex under basal or stress conditions, and levels of the target kinase Hog1.</p

Hsp90 genetic interactions identified in six screens.

<p>Hsp90 genetic interactions identified in six screens.</p

Hsp90 kinase genetic interaction network.

<p>Of the 226 interactions, 34 are with kinases. Kinases are color-coded depending on their degree of connectivity, ranging from grey for one connection to orange for five connections. Kinases and test conditions (black squares) are connected with each other via edges. While the caspofungin screen shared only one of its kinase interactors (<i>CKB2</i>) with another screen, every other screen shared half or more of its interactors with another screen. For six kinases (diamonds), protein levels were measured upon Hsp90 depletion.</p

Model of high- and low-connectivity interactors identified in this screen that either modify the Hsp90/Cdc37 complex or are affected by it.

<p>The high connectivity interactors (red) modulate gene expression, protein levels, or phosphorylation of Hsp90 and Cdc37, while the chaperone complex regulates gene expression, protein levels or activation of low-connectivity interactors (blue).</p

Hsp90 client protein Cna1 retains stability upon depletion of Sgt1, though Sgt1 is required for Cna1 activation.

<p>(A) Cna1 retains stability upon depletion of Sgt1. Sgt1 levels were reduced by growth overnight in 20 µg/ml doxycycline, followed by subculture in fresh medium with 20 µg/ml doxycycline and growth until mid-log phase. First panel, immune blot analysis of Sgt1 levels (50 µg protein loaded per well); and second panel, immune blot analysis of Cna1-TAP levels (50 µg protein loaded per well). Actin was used as a loading control. (B) Sgt1 is required for Cna1 activation in response to fluconazole or micafungin. Transcript levels of a Cna1-dependent gene, <i>UTR2</i>, were measured for the wild-type (<i>SGT1/SGT1</i>) or <i>tetO-SGT1/sgt1</i>Δ strains by quantitative RT-PCR after growth in rich medium at 30°C with or without 20 µg/ml doxycycline (DOX), 16 µg/ml fluconazole (FL), and 30 ng/ml micafungin (MF), as indicated. <i>UTR2</i> transcript levels were normalized to <i>GPD1</i>. Data are means ± standard deviations for triplicate samples.</p

Genetic depletion of Sgt1 induces filamentation, wrinkly colony morphology and invasive growth.

<p>(A) Genetic depletion of Sgt1 induces filamentation. Sgt1 levels were reduced by growth overnight in 20 µg/ml doxycycline, followed by subculture in fresh medium with 20 µg/ml doxycycline. (B) Genetic depletion of Sgt1 in <i>the tetO-SGT1/sgt1</i>Δ strain leads to wrinkly colony morphology and invasive growth. Cells were plated on YPD agar and grown at 37°C for 3 days. Colonies were washed from the plates with H₂O. (C) The <i>tetO-SGT1/sgt1</i>Δ strain has lower <i>SGT1</i> transcript levels compared to wild type in the absence of doxycycline (** indicates <i>P<</i>0.01, ANOVA), and <i>SGT1</i> levels are completely depleted upon treatment with doxycycline (* indicates <i>P<</i>0.05, ANOVA). Wild type, <i>SGT1/sgt11</i>Δ, or <i>tetO-SGT1/sgt1</i>Δ cells grown overnight in 20 µg/ml doxycycline, followed by subculture in fresh medium with 20 µg/ml doxycycline and grown until mid-log phase, as indicated. <i>SGT1</i> transcript levels were normalized to <i>GPD1</i>. Data are means ± standard deviations for triplicate samples.</p

The Hsp90 Co-Chaperone Sgt1 Governs <em>Candida albicans</em> Morphogenesis and Drug Resistance

<div><p>The molecular chaperone Hsp90 orchestrates regulatory circuitry governing fungal morphogenesis, biofilm development, drug resistance, and virulence. Hsp90 functions in concert with co-chaperones to regulate stability and activation of client proteins, many of which are signal transducers. Here, we characterize the first Hsp90 co-chaperone in the leading human fungal pathogen, <em>Candida albicans</em>. We demonstrate that Sgt1 physically interacts with Hsp90, and that it governs <em>C. albicans</em> morphogenesis and drug resistance. Genetic depletion of Sgt1 phenocopies depletion of Hsp90, inducing yeast to filament morphogenesis and invasive growth. Sgt1 governs these traits by bridging two morphogenetic regulators: Hsp90 and the adenylyl cyclase of the cAMP-PKA signaling cascade, Cyr1. Sgt1 physically interacts with Cyr1, and depletion of either Sgt1 or Hsp90 activates cAMP-PKA signaling, revealing the elusive link between Hsp90 and the PKA signaling cascade. Sgt1 also mediates tolerance and resistance to the two most widely deployed classes of antifungal drugs, azoles and echinocandins. Depletion of Sgt1 abrogates basal tolerance and acquired resistance to azoles, which target the cell membrane. Depletion of Sgt1 also abrogates tolerance and resistance to echinocandins, which target the cell wall, and renders echinocandins fungicidal. Though Sgt1 and Hsp90 have a conserved impact on drug resistance, the underlying mechanisms are distinct. Depletion of Hsp90 destabilizes the client protein calcineurin, thereby blocking crucial responses to drug-induced stress; in contrast, depletion of Sgt1 does not destabilize calcineurin, but blocks calcineurin activation in response to drug-induced stress. Sgt1 influences not only morphogenesis and drug resistance, but also virulence, as genetic depletion of <em>C. albicans</em> Sgt1 leads to reduced kidney fungal burden in a murine model of systemic infection. Thus, our characterization of the first Hsp90 co-chaperone in a fungal pathogen establishes <em>C. albicans</em> Sgt1 as a global regulator of morphogenesis and drug resistance, providing a new target for treatment of life-threatening fungal infections.</p> </div

Sgt1 enables basal tolerance and <i>erg3</i>-mediated resistance to the azoles.

<p>(A) Reduced levels of Sgt1 renders cells sensitive to fluconazole in minimum inhibitory concentration (MIC) assays. Assays were performed in YPD medium with a gradient of fluconazole from 0 to 256 µg/ml, in two-fold dilutions, with or without a fixed concentration of 20 µg/ml doxycycline (DOX), as indicated. Growth was measured by absorbance at 600 nm after 48 hours at 30°C. Optical densities were averaged for duplicate measurements and normalized relative to the no fluconazole control. Data was quantitatively displayed with colour using Treeview (see colour bar). (B) Reduced levels of Sgt1 renders azole-resistant <i>erg3</i> mutants sensitive to fluconazole in MIC assays. Assays were performed in YPD medium with a gradient of fluconazole from 0 to 256 µg/ml, in two-fold dilutions, with a fixed concentration of 20 µg/ml doxycycline, as indicated. Growth was measured after 72 hours at 30°C. Data was analyzed as in part A. (C) Cells with reduced levels of Sgt1 remain viable after exposure to fluconazole. MIC assays were performed in YPD medium with a gradient of fluconazole from 0 to 256 µg/ml, in two-fold dilutions, with or without a fixed concentration of 20 µg/ml doxycycline, as indicated. Assays were grown for 48 hours at 30°C, and cells from the MIC assays were spotted onto YPD medium and incubated at 30°C for 48 hours before plates were photographed.</p