Contribution of Fdh3 and Glr1 to Glutathione Redox State, Stress Adaptation and Virulence in Candida albicans

Acknowledgments: We thank Aaron Mitchell and Dominique Sanglard for providing the C. albicans protein kinase and transposon mutant libraries, and Louise Walker for the strain CAMY203.Peer reviewedPublisher PD

From START to FINISH : the influence of osmotic stress on the cell cycle

Peer reviewedPublisher PD

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

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

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

The metabolic background is a global player in Saccharomyces gene expression epistasis

The regulation of gene expression in response to nutrient availability is fundamental to the genotype–phenotype relationship. The metabolic–genetic make-up of the cell, as reflected in auxotrophy, is hence likely to be a determinant of gene expression. Here, we address the importance of the metabolic–genetic background by monitoring transcriptome, proteome and metabolome in a repertoire of 16 Saccharomyces cerevisiae laboratory backgrounds, combinatorially perturbed in histidine, leucine, methionine and uracil biosynthesis. The metabolic background affected up to 85% of the coding genome. Suggesting widespread confounding, these transcriptional changes show, on average, 83% overlap between unrelated auxotrophs and 35% with previously published transcriptomes generated for non-metabolic gene knockouts. Background-dependent gene expression correlated with metabolic flux and acted, predominantly through masking or suppression, on 88% of transcriptional interactions epistatically. As a consequence, the deletion of the same metabolic gene in a different background could provoke an entirely different transcriptional response. Propagating to the proteome and scaling up at the metabolome, metabolic background dependencies reveal the prevalence of metabolism-dependent epistasis at all regulatory levels. Urging a fundamental change of the prevailing laboratory practice of using auxotrophs and nutrient supplemented media, these results reveal epistatic intertwining of metabolism with gene expression on the genomic scale

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

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