Integrative Model of Oxidative Stress Adaptation in the Fungal Pathogen Candida albicans

Acknowledgments We are grateful to the Ian Fraser Cytometry Centre and our Mass Spetrometry and qPCR Facilities for help with the flow cytometry, glutathione and qRT-PCR assays, respectively. We also thank our many colleagues in the CRISP Consortium and in the medical mycology and systems biology communities for insightful discussions. Funding: This work was supported by the CRISP project (Combinatorial Responses In Stress Pathways), which was funded by the UK Biotechnology and Biological Research Council (www.bbsrc.ac.uk): AJPB, KH, CG, ADM, NARG, MT, MCR. (Research Grants; BB/F00513X/1, BB/F005210/1-2). AJPB and JQ received additional support from the BBSRC (Research Grants; BB/K016393/1; BB/K017365/1). NARG and AJPB were also supported by the Wellcome Trust (www.wellcome.ac.uk), (Grants: 080088; 097377). AJPB was also supported by the European Research Council (http://erc.europa.eu/), (STRIFE Advanced Grant; ERC-2009-AdG-249793). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

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

Temporal changes in the levels of (a) active Cap1 (Cap1^A) and (b) <i>TRR1</i> mRNA levels following exposure to 5 mM H₂O₂.

<p>Model simulations are represented by black solid lines (left hand Y-axes), and experimental measurements by blue boxes (three independent experiments; right hand Y-axis). Relative <i>TRR1</i> mRNA levels were measured relative to the internal <i>ACT1</i> mRNA control. Standard deviation was calculated and is shown in the figure.</p

Comprehensive reaction network model of the oxidative stress response in <i>C</i>. <i>albicans</i>.

<p>A description of the modules and sub-modules and the list of components considered in this study are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137750#pone.0137750.t001" target="_blank">Table 1</a>. The biochemical reactions and system components are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137750#pone.0137750.s004" target="_blank">S1</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137750#pone.0137750.s005" target="_blank">S2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137750#pone.0137750.s008" target="_blank">S5</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137750#pone.0137750.s009" target="_blank">S6</a> Tables. In addition to the biochemical reactions that are marked in this figure, all components of the model are also assumed to undergo a first order decay. See <i>Model Construction</i> for further details.</p

The model accurately predicts the temporal protection provided by oxidative stress adaptation.

<p><b>(a)</b> Representation of the timing of the sequential stresses applied in the experiment. The white arrow represents the initial 0.4mM mM H₂O₂ stress (T1), whilst the colored arrows represent the addition of the second 20 mM H₂O₂ stress (T2) at the following times after the first stress: 0 min (black arrow); 60 min (red arrow); 120 min (grey arrow); 180 min (green arrow); 240 min (blue arrow). <b>(b)</b> The predicted dynamics of intracellular H₂O₂ levels (H₂O₂^In), and <b>(c)</b> redox potential (ΔE) after imposition of the second 20 mM H₂O₂ stress at: 0 min (black); 60 min (red); 180 min (green); 240 min (blue). <b>(d)</b> Experimentally measured survival of <i>C</i>. <i>albicans</i> cells after exposure to the sequential stresses when T2 was: 0 min (black); 60 min (red); 180 min (green); 240 min (blue). Colony forming units (CFU) for stressed cells were measured relative to the untreated control cells. The data represent mean and standard deviation values from three independent experiments: *, P<0.05; **, P<0.01.</p

Temporal changes in the levels of stressor, glutathione, glutathione disulphide and thioredoxin following exposure to 5 mM H₂O₂.

<p>Model simulations (black solid lines) are compared with the corresponding measurements based on three independent experiments (blue boxes). <b>(a)</b> Extracellular hydrogen peroxide (H₂O₂^Ex): left hand Y-axis, simulated levels (mM); right hand Y-axis, experimental measurements (percent of initial value). <b>(b)</b> Glutathione (GSH) left hand Y-axis, simulated levels (mM); right hand Y-axis, experimental measurements (mM). <b>(c)</b> Glutathione disulphide (GSSG) left hand Y-axis, simulated levels (mM); right hand Y-axis, experimental measurements (mM). <b>(d)</b> Simulated levels of the oxidised form of thioredoxin (Trx1^Ox). Experimental errors represent standard deviations from at least three measurements.</p

Catalase (Cat1) inactivation shifts the dose response curve to lower H₂O₂ concentrations.

<p>Using qRT-PCR, relative <i>CAP1</i> and <i>TRR1</i> mRNA levels were measured relative to the internal <i>ACT1</i> mRNA control in <i>C</i>. <i>albicans</i> cells exposed the same range of H₂O₂ concentrations examined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137750#pone.0137750.g004" target="_blank">Fig 4</a> (grey scale, top left): upper panel, wt (wild type, CA372); lower panel, <i>cat1</i> (CA1864) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137750#pone.0137750.s010" target="_blank">S7 Table</a>). The data is representative of three independent experiments and the standard deviation was calculated.</p

Dose-dependent response of Cap1-dependent genes following exposure to different H₂O₂ concentrations.

<p><b>(a)</b> Data from Enjalbert <i>et al</i>. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137750#pone.0137750.ref054" target="_blank">54</a>] showing GFP expression levels from <i>CAT1-</i>, <i>TTR1</i>-, and <i>TRX1-GFP</i> reporters in <i>C</i>. <i>albicans</i> cells exposed to a range of H₂O₂ concentrations (grey scale, bottom right). GFP intensities are expressed in absorbance units. <b>(b)</b> Data from this study showing the levels of <i>CAP1</i>, <i>CAT1</i> and <i>TTR1</i> mRNAs in <i>C</i>. <i>albicans</i> cells exposed the same range of H₂O₂ concentrations. Relative mRNA levels were measured by qRT-PCR relative to the internal <i>ACT1</i> mRNA control. We show data of three independent experiments and the corresponding SD (t-test). <b>(c)</b> Simulation results for <i>TRR1</i> mRNA levels (nM) after exposure to different H₂O₂ concentrations, obtained using oxidative stress models that lack (2 Cap1 Forms) or include the third conceptual form of Cap1 (3 Cap1 Forms). <b>(d)</b> Proposed model of Cap1 regulation in <i>C</i>. <i>albicans</i>.</p

Contributions of the three antioxidant systems to H₂O₂ detoxification and resistance.

<p><b>(a)</b> Experimental measurements of hydrogen peroxide levels in the medium (H₂O₂^Ex) of mid-exponential <i>C</i>. <i>albicans</i> cultures following exposure to 5 mM H₂O₂, relative to this starting H₂O₂^Ex concentration (%): wt, <i>C</i>. <i>albicans</i> wild type (CA372); <i>cap1</i> (JC842); <i>hog1</i> (JC45); <i>cat1</i> (CA1864); <i>trx1</i> (JC677); <i>glr1</i> (<i>glr1</i>Δ/ <i>glr1</i>Δ) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137750#pone.0137750.s010" target="_blank">S7 Table</a>). <b>(b)</b> Model simulations of H₂O₂^Ex levels following addition of 5 mM H₂O₂ to <i>C</i>. <i>albicans</i> cultures. <b>(c)</b> Growth of serial ten-fold dilutions of <i>C</i>. <i>albicans</i> wild type, <i>cat1</i>, <i>cap1</i>, <i>hog1</i>, <i>glr1</i> and <i>trx1</i> cells on YPD plates containing H₂O₂ after 48 h at 30°C.</p