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

Virulence of <i>C</i>. <i>albicans GLR1</i> and <i>FDH3</i> mutants in the <i>G</i>. <i>mellonella</i> infection model.

<p>Kaplan-Meier plots of <i>G</i>. <i>mellonella</i> survival after injection with <i>C</i>. <i>albicans</i>. <b>(A)</b> Analysis of deletion mutants using a dose of 2.5x10⁵<i>C</i>. <i>albicans</i> cells/larva: wild type (CPK05); <i>glr1</i>Δ (CKS10); <i>glr1</i>Δ+GLR1 (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> Analysis of overexpression mutants using a lower dose of 1x10⁴<i>C</i>. <i>albicans</i> cells/larva: WT+DOX, <i>tetON-empty</i> (CAMY203); GLR1+DOX, <i>tetON-GLR1</i> (ATT6); FDH3+DOX, <i>tetON-FDH3</i> (ATT7).</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

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

Lack of Fdh3 or Glr1 affects GSSG and GSNO detoxification and the glutathione redox potential.

<p><b>(A)</b> GSSG detoxification by protein extracts from wild type (CPK05), <i>glr1</i>Δ (CKS10), <i>glr1</i>Δ+<i>GLR1</i> (CKS31), <i>fdh3</i>Δ (ATT1) and <i>fdh3</i>Δ+<i>FDH3</i> (ATT4) cells. <b>(B)</b> GSNO detoxification by protein extracts from wild type (CPK05), <i>glr1</i>Δ (CKS10), <i>glr1</i>Δ+<i>GLR1</i> (CKS31), <i>fdh3</i>Δ (ATT1) and <i>fdh3</i>Δ+<i>FDH3</i> (ATT4) cells. <b>(C)</b> Glutathione redox potential for wild type (CPK05), <i>glr1</i>Δ (CKS10), <i>glr1</i>Δ+GLR1 (CKS31), <i>fdh3</i>Δ (ATT1) and <i>fdh3</i>Δ+<i>FDH3</i> (ATT4) strains.</p

Effect of <i>FDH3</i> and <i>GLR1</i> deletion on basal gene expression.

<p>Quantification of <i>FDH3</i> and <i>GLR1</i> mRNA levels by qRT-PCR, relative to the internal <i>ACT1</i> mRNA control and normalised to wild type cells: wild type (BWP17); <i>fdh3</i>Δ (ATT1); <i>glr1</i>Δ (CKS10).</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

Perturbation of Redox Potential (ΔE) is a reasonable proxy for oxidative stress sensitivity.

<p><b>(a)</b> Simulated changes in ΔE in <i>C</i>. <i>albicans</i> cells following exposure to 5 mM H₂O₂: wt, wild type (CA372); <i>cap1</i> (JC842); <i>hog1</i> (JC45); <i>cat1</i> (CA1864) and <i>cap1 hog1</i> (JC118) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137750#pone.0137750.s010" target="_blank">S7 Table</a>). The dotted line represents -180 mV, above which cells are more likely to enter oxidant-driven cell death pathways [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137750#pone.0137750.ref058" target="_blank">58</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137750#pone.0137750.ref059" target="_blank">59</a>]. <b>(b)</b> Experimental determination of percentage survival following exposure of the <i>C</i>. <i>albicans</i> strains to 5 mM H₂O₂: *, P<0.05; **, P<0.01; ***, P<0.001, using an Unpaired t-test.</p

Cellular modules/sub-modules of the comprehensive reaction network model and their components.

<p>Cellular modules/sub-modules of the comprehensive reaction network model and their components.</p