Time course activity of cell cycle components upon application of 1 M NaCl at early S phase.

<p>The left vertical axis refers to the concentrations of total Sic1, SBF/MBF, Swe1, Cdc28-Clb2, Cdc28-Clb5, and Hog1PP and the right vertical axis refers to the concentration of the Hsl1-Hsl7 complex. (A) A wild type untreated cell, (B) 1 M NaCl applied during early S phase (at t = 45 min) to a wild type cell causes the cell cycle to last about 76 minutes longer compared to the wild type untreated cell. (C) 1 M NaCl applied to a Δ<i>swe1</i> cell; in this case the cell cycle duration is 62 minutes longer than in an untreated Δ<i>swe1</i> cell. (D) The deletion of Sic1 does not cancel the delay caused by Hog1PP activity. 1 M NaCl applied to a Δ<i>sic1</i> cell prolongs the cell cycle around 52 minutes compared to a Δ<i>sic1</i> untreated cell.</p

The HOG MAPK network rescues the mitotic exit defect of MEN mutants.

<p>(A) A <i>cdc15</i> cell is arrested in M phase and cannot divide. (B) Application of 0.4 M NaCl stimulates the <i>cdc15</i> cell to go through cell division. (C) The <i>cdc14</i> cell can go through the cell division in the presence of 0.4 M NaCl. (D) Removing the interaction of Hog1PP with <i>CLB2</i> does not cancel the cell division of the <i>cdc15</i> cell in the presence of osmotic stress. Note that the <i>cdc15</i> cell upon osmotic stress is able to finish its current cell cycle but gets arrested in the next G2/M phase. (E) The <i>cdc15</i> cell, in which the interaction of Sic1 with Hog1PP is blocked, cannot finish its cell cycle and is arrested in M phase.</p

Application of osmotic stress during late S phase or early G2/M phase causes DNA re-replication.

<p>(A) Time course activity of the cell cycle components for the wild type untreated cell. (B) 1 M NaCl applied at minute 76. Activity of Hog1PP causes downregulation of Cdc28-Clb5. In addition, the level of Cdc6 slightly increases when Cdc28-Clb5 activity is reduced by Hog1PP (see inset). Then, after Hog1PP returns to its basal level, Clb5 starts increasing again. The downregulation, following by an upregulation of Cdc28-Clb5 can lead to DNA re-replication. (C) Overexpression of <i>CLB5</i>, by simulating induction of <i>CLB5</i> transcription from the GAL1 promoter, inhibits the DNA re-replication. (D) Blocking the interaction of Sic1 with Hog1PP also hinders the DNA re-replication in the presence of 1 M NaCl.</p

Assessing the role of two key mechanisms responsible for the cell adaptation to osmotic stress during the G1 phase.

<p>The x-axis represents the time point of application of stress, whereas the y-axis illustrates the corresponding arrest duration. Blocking the interaction of Sic1 with Hog1PP, reduces the arrest duration significantly along the G1 phase (red crosses).</p

Dose-dependent arrest duration following the imposition of osmotic stress at different stages of the cell cycle.

<p>The x-axis represents the time point of application of stress, whereas the y-axis illustrates the corresponding arrest duration. Different colours demonstrate various doses of the stress, ranging from 0.4 M NaCl to 1 M NaCl. During the G1 phase and the S phase, higher doses of stress cause longer cell cycle arrests, while the acceleration of the exit from mitosis is dose independent.</p

Differential sensitivities of <i>C</i>. <i>albicans fdh3</i>Δ and <i>glr1</i>Δ cells to hydrogen peroxide, nitric oxide and formaldehyde.

<p><b>(A)</b> Sensitivity to hydrogen peroxide (7.5 mM H₂O₂) and formaldehyde (5 mM CH₂O): wild type (CPK05); <i>glr1</i>Δ (CKS10), <i>glr1</i>Δ+<i>GLR1</i> (CKS31), <i>fdh3</i>Δ (ATT1); <i>fdh3</i>Δ+<i>FDH3</i> (ATT4) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126940#pone.0126940.t001" target="_blank">Table 1</a>). <b>(B)</b> Dose-dependent sensitivity to formaldehyde: wild type (CPK05); <i>fdh3</i>Δ (ATT1); <i>fdh3</i>Δ+<i>FDH3</i> (ATT4). <b>(C)</b> Differences in adaptation (inflection) time after nitrosative stress (2.5 mM DPTA NONOate): wild type (CPK05); <i>glr1</i>Δ (CKS10); <i>glr1</i>Δ+<i>GLR1</i> (CKS31); <i>fdh3</i>Δ (ATT1); <i>fdh3</i>Δ+<i>FDH3</i> (ATT4).</p

<i>C</i>. <i>albicans</i> Glr1 and Fdh3 belong to evolutionary conserved families of glutathione reductases (GRs) and S-nitroso-glutathione reductases (GSNORs), respectively.

<p><b>(A)</b> The functional domains of GSH-dependent formaldehyde dehydrogenases class III (GSNORs) and NADPH-dependent glutathione reductases (GRs). GSNORs harbour a catalytic domain (ADH), an NAD(H) binding domain, and a dimerization domain. GRs have an NADH- and FAD-binding domains and a dimerization domain. <b>(B)</b> Phylogenetic tree of GSNOR- and GR-related proteins generated using ClustalW: homologs are presented from <i>Candida albicans</i> (CaFdh3, CaGlr1), <i>Saccharomyces cerevisiae</i> (ScSfa1, ScGlr1), <i>Schizosaccharomyces pombe</i> (SpSPCC13B11.04c, SpPgr1), <i>Mus musculus</i> (MsAdh5; MsGsr1), <i>Homo sapiens</i> (HsAdh5; HsGsr), <i>Drosophila melanogaster</i> (DmFdh) and <i>Caenorhabditis elegans</i> (CeH24K24.3; CeGsr-1). Structures are presented for human liver ChiChi alcohol dehydrogenase (protein data bank (pdb) accession code 1TEH; a GSNOR that has 65% sequence identity to CaFdh3p), and <i>S</i>. <i>cerevisiae</i> Glr1 (pdb accession code 2HQM; a GR with 66% sequence identity to CaGlr1p). Structure representations were made with PyMOL (<a href="http://www.pymol.org" target="_blank">http://www.pymol.org</a>).</p

Deletion or overexpression of <i>GLR1</i> or <i>FDH3</i> alters the ability of <i>C</i>. <i>albicans</i> to kill macrophages.

<p><i>C</i>. <i>albicans deletion</i> (Δ) and overexpression (O/E) mutants (1x10⁶ cells) were co-incubated with RAW264.7 macrophages (2x10⁵) for 3 h. The proportion of killed macrophages was determined following trypan blue staining: wild type (CPK05), <i>glr1</i>Δ (CKS10), <i>glr1</i>Δ+<i>GLR1</i> (CKS31), <i>fdh3</i>Δ (ATT1); <i>fdh3</i>Δ+<i>FDH3</i> (ATT4); WT+DOX, <i>tetON-empty</i> (CAMY203); GLR1+DOX, <i>tetON-GLR1</i> (ATT6); FDH3+DOX, <i>tetON-FDH3</i> (ATT7).</p

Impact of <i>FDH3</i> and <i>GLR1</i> deletion on transcript levels in respose to formaldehyde, oxidative or nitrosative stress.

<p>Transcript levels were quantified by qRT-PCR, relative to the internal <i>ACT1</i> mRNA control after 10 min of stress treatment and normalised to untreated wild type cells: wild type (CPK05); <i>fdh3</i>Δ (ATT1); <i>glr1</i>Δ (CKS10). Stresses were 2.5 mM CySNO (NS), 5 mM CH₂O or 5 mM H₂O₂ (XS). Gene expression was assayed for the following genes: (A) <i>GLR1</i>, (B) <i>FDH3</i>, (C) <i>TRX1</i>, (D) <i>PDI1</i>.</p

Predicted roles for Fdh3 and Glr1 in <i>C. albicans</i>.

<p><b>(A)</b> Predicted roles for Fdh3 and Glr1 in GSNO and GSSG detoxification. <b>A</b> shows the working hypothesis of the major enzymes involved in the detoxification of GSSG (glutathione disulphide) and GSNO (S-nitrosoglutathione). When glutathione (GSH) is oxidised via H₂O₂ to GSSG, GSSG can be reduced with the help of the NADPH-dependent glutathione reductase (GR). We predict that the glutathione reductase of <i>Candida albicans</i> is <i>GLR1</i>. When GSH is exposed to NO, GSH is S-nitrosylated to GSNO. We predict that the S-nitrosoglutathione reductase (GSNOR) of <i>Candida albicans</i> is <i>FDH3</i>. <b>(B)</b> Predicted role for Fdh3 in formaldehyde detoxification. <b>B</b> shows the second enzymatic function of GSNOR the detoxification of formaldehyde. Formaldehyde reacts with glutathione (GSH) to form S-(hydroxmethyl)glutathione which then gets converted by Fdh3 and NAD+ to S-(formyl)glutathione.</p