Impact of MAP kinase cross-talk upon stress resistance during thermal adaptation.

<p>Cek1 signalling is required for the Calcofluor White resistance of <i>hog1</i> cells at high temperatures. MIC assays were performed in YPD medium supplemented with different concentrations of NaCl, Calcofluor White or Congo Red. Plates were incubated statically at 25°C, 30°C, 37°C and 42°C for 48 hours. For each strain, optical densities were averaged for duplicate measurements and growth is quantitatively displayed with colour as indicated with the colour bar. Data are representative of three biological replicates. WT, wild type (NGY152: <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#ppat-1003069-t001" target="_blank">Table 1</a>).</p

Hsp90 depletion decreases stress resistance.

<p>(A) Impact of Hsp90 depletion upon stress resistance (CFUs) was measured following doxycycline treatment of <i>C. albicans</i> wild-type and <i>tetO-HSP90</i> cells (SN95 and CaLC1411: <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#ppat-1003069-t001" target="_blank">Table 1</a>): white bars, wild type, no doxycycline; light grey bars, wild type plus 20 µg/ml doxycycline; dark grey bars, <i>tetO-HSP90</i> no doxycycline; black bars, <i>tetO-HSP90</i> plus 20 µg/ml doxycycline; CFW, 100 µg/ml Calcofluor White; CR, 100 µg/ml Congo Red; HS, 30°C–42°C heat shock; 5 mM H₂O₂; 1 M NaCl. (B) Impact of Hsp90 inhibition with the pharmacological inhibitor geldanamycin. Cells were grown for 7 hours in the absence or presence of 10 µM geldanamycin and subjected to the same stresses as in (A). CFUs determined from untreated cells. All data are the means from three independent assays: ** paired, two-tailed t-test, p<0.01.</p

Hsp90 depletion affects cell wall architecture.

<p><i>C. albicans</i> wild-type cells (SN95), and <i>tetO-HSP90</i> (CaLC1411: <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#ppat-1003069-t001" target="_blank">Table 1</a>) were treated with 0 or 20 µg/ml doxycycline for seven hours. Chitin levels were assayed by Calcofluor White staining, scale bars are 10 µm (A) and quantification of fluorescence levels (B). Data represent means from fifty cells: ** paired, two-tailed t-test, p<0.01. The architecture of the cell wall was examined by transmission electron microscopy where scale bars are 200 µm (C), and the thickness of the cell wall quantified in n = 30 cells (D): ** paired, two-tailed t-test, p<0.01.</p

Hsp90 coordinates the activities of multiple signalling pathways that contribute to thermotolerance in <i>C. albicans</i> - a model.

<p>Hsf1 activation is required for thermotolerance <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#ppat.1003069-Nicholls3" target="_blank">[42]</a>. Hog1, Mkc1 and Cek1 signalling are also required for thermotolerance (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#ppat-1003069-g003" target="_blank">Figure 3</a>), but these MAP kinases are not essential for Hsf1 phosphorylation (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#ppat-1003069-g004" target="_blank">Figure 4</a>). Instead, these pathways promote thermotolerance in part via cell wall remodelling <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#ppat.1003069-Eisman1" target="_blank">[81]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#ppat.1003069-NavarroGarca1" target="_blank">[90]</a>. Hsp90 coordinates much of this activity. Hsf1 (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#ppat-1003069-g001" target="_blank">Figures 1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#ppat-1003069-g002" target="_blank">2</a>), Hog1, Mkc1 and Cek1 (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#ppat-1003069-g010" target="_blank">Figure 10</a>) are all Hsp90 client proteins <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#ppat.1003069-Diezmann1" target="_blank">[62]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#ppat.1003069-LaFayette1" target="_blank">[86]</a>. Changes in ambient temperature affect interactions between Hsp90 and Hsf1 (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#ppat-1003069-g002" target="_blank">Figure 2</a>), and probably affect Hsp90 interactions with Hog1, Mkc1 and Cek1 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#ppat.1003069-Leach1" target="_blank">[49]</a> thereby modulating the activities of these signalling pathways and their inputs to thermal adaptation. Increases in ambient temperature activate Hsf1, thereby inducing the expression of protein chaperones (HSPs) including Hsp90, which promotes thermal adaptation in the shorter term. Hsp90 then down-regulates Hsf1 and modulates Mkc1, Hog1 and Cek1 signalling, which in the longer term influences cell wall architecture (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#ppat-1003069-g011" target="_blank">Figure 11</a>), leading to the thermotolerance of <i>C. albicans</i>.</p

<i>C. albicans</i> strains.

<p><i>C. albicans</i> strains.</p

Hsp90 depletion affects MAP kinase signalling in the presence and absence of stress.

<p><i>C. albicans</i> wild-type and <i>tetO-HSP90</i> cells (SN95 and CaLC1411: <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#ppat-1003069-t001" target="_blank">Table 1</a>) were treated with 0 or 20 µg/ml doxycycline for seven hours. Mkc1, Cek1 and Hog1 phosphorylation levels were then assayed by western analysis in unstressed cells or cells treated as follows: (A) 30 minute 30°C–42°C heat shock; (B) 30 minutes with 100 µg/ml Calcofluor White (CFW); (C) 10 minutes with 5 mM H₂O₂; or (D) 12 minutes with 1 M NaCl. Total Mkc1 levels were assayed using Mkc1-6xHis-FLAG tagged cells in SN95 (CaLC681) and <i>tetO-HSP90</i> (caLC648) cells (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#ppat-1003069-t001" target="_blank">Table 1</a>). Hsp90 levels and total kinase levels for Hog1 were also examined by western blotting relative to the internal Act1 loading control.</p

Cross-talk between MAP kinase pathways during heat shock.

<p>Differential MAP kinase activation in response to a 30°C–42°C heat shock in MAP kinase mutants. (A) Activation of Hog1, Mkc1 and Cek1 was determined in an <i>mkc1</i>Δ mutant relative to the internal Act1 control. (B) Activation of Hog1, Mkc1 and Cek1 was assayed in a <i>cek1</i>Δ mutant relative to the internal Act1 control. (C) Activation of Hog1, Mkc1 and Cek1 was determined in a <i>hog1</i>Δ mutant relative to the internal Act1 control.</p

The cross-talk between thermal adaptation and cell wall stress resistance is not mediated via stress cross-protection.

<p>(A) Stress cross-protection in <i>C. albicans</i> wild-type cells (NGY152: <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#ppat-1003069-t001" target="_blank">Table 1</a>) was not observed for cells pre-treated with a heat stress and then subjected to cell wall stress, but was observed for cells exposed to a secondary oxidative stress. The data represent cell survival after exposure to a 30°C–42°C heat shock followed by a subsequent cell wall (CFW, CR), osmotic (NaCl) or peroxide (H₂O₂) stress (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#s2" target="_blank">Materials and Methods</a>) and the data are expressed relative to unstressed cells (dark bars). Control cells (grey bars), were not exposed to the prior 30°C–42°C heat shock. (B) The reciprocal assay was performed, whereby <i>C. albicans</i> wild type cells were exposed to a prior stress (cell wall: CFW, CR; osmotic: NaCl; peroxide: H₂O₂) followed by a 30°C–42°C heat shock (dark bars). These data are expressed relative to unstressed cells. Control cells (grey bars) correspond to cells exposed only to the 30°C–42°C heat shock. (C) Wild type, <i>hog1</i>Δ (JC50) or <i>cap1</i>Δ cells (JC128: <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#ppat-1003069-t001" target="_blank">Table 1</a>) were pre-treated with a 30°C–42°C heat shock, followed by a H₂O₂ stress (dark bars). Control cells (grey bars) were not exposed to the 30°C–42°C heat shock. The data represent the level of survival compared to unstressed cells. All data are the means from three independent assays: ** paired, two-tailed t-test, p<0.01.</p

Key MAP kinase signalling pathways contribute to thermal adaptation in <i>C. albicans</i>.

<p>Figure depicting protein kinase mutants screened for temperature phenotypes. Temperature sensitivity was assayed by monitoring the growth of <i>C. albicans</i> mutants following a 30°C–42°C heat shock. Triplicate primary screens examined transposon mutants <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#ppat.1003069-Nobile1" target="_blank">[57]</a>, and triplicate secondary screens tested <i>C. albicans</i> null mutants (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#ppat-1003069-t001" target="_blank">Table 1</a>). Temperature resistant kinases are displayed in the blue column, kinases with no apparent role in thermal adaptation are displayed in the pale columns, and temperature sensitive kinases are present in the pink (10–19%) to red (>20%) columns.</p

Hsp90 depletion destabilises Cek1.

<p><i>C. albicans CEK1-TAP</i> (CaLC2287) and <i>tetO-HSP90 CEK1-TAP</i> (CaLC2288: <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003069#ppat-1003069-t001" target="_blank">Table 1</a>) cells were treated with 0 or 20 µg/ml doxycycline for seven hours and subjected to western analysis. Decreased Cek1-TAP levels were observed following Hsp90 depletion in unstressed cells and in cells treated as follows: (A) 30 minute 30°C–42°C heat shock; or (B) 30 minutes with 100 µg/ml Calcofluor White (CFW). Hsp90 protein levels were examined confirming significant depletion following doxycycline treatment of <i>tetO-HSP90</i> cells. Actin served as the internal loading control.</p