Fungal iron scavenging competes with mammalian nutritional immunity.

<p>Pathogenic fungi employ different combinations of evolutionarily related strategies to acquire iron in the limiting microenvironments of the host. The figure focuses on those fungal species for which data are currently available. See text for details. Hb: haemoglobin, RIA: reductive iron acquisition, <i>Afum</i>: <i>A. fumigatus</i>, <i>Calb</i>: <i>C. albicans</i>, <i>Cneo</i>: <i>Cr. neoformans</i>, <i>Hcap</i>: <i>H. capsulatum</i>, <i>Rory</i>: <i>R. oryzae</i>.</p

Validation of <i>Clox</i> cassettes for multi-marker gene disruption.

<p>The <i>LHL</i> and <i>URA3-Clox</i> cassettes were used to generate a homozygous <i>ade2Δ/ade2Δ</i> mutation in <i>C. albicans</i> RM1000. (A) Confirmation of the expected auxotrophic requirements for wild type (<i>ADE2/ADE2</i>), heterozygous (<i>ade2Δ::LHL/ADE2</i>), unresolved homozygous (<i>ade2Δ::LHL/ade2Δ::URA3-Clox</i>) and resolved homozygous mutants (<i>ade2Δ::loxP/ade2Δ::loxP</i>). Growth media are specified on the right: w/o, without a specific supplement. (B) PCR confirmation of the genotypes for these mutants using primers specific for each allele (specified on the left of each panel). PCR product lengths are given on the right of each panel. <i>ACT1</i> was used as a positive control, and a no-DNA control was included (Control).</p

Validation of <i>NAT1-Clox</i> for single marker recycling in a prototrophic clinical isolate.

<p><i>NAT1-Clox</i> was used to generate a homozygous <i>ade2Δ/ade2Δ</i> mutation in <i>C. albicans</i> SC5134. (A) Confirmation of the expected phenotypes for wild type (<i>ADE2/ADE2</i>), unresolved heterozygous (<i>ADE2/ade2Δ::NAT1-Clox</i>), resolved heterozygous (<i>ADE2/ade2Δ::loxP</i>), unresolved homozygous (<i>ade2Δ::loxP/ade2Δ::NAT1-Clox</i>) and resolved homozygous mutants (<i>ade2Δ::loxP/ade2Δ::loxP</i>). Growth media are specified on the right: w/o, without a specific supplement; nou, nourseothricin. (B) PCR confirmation of the genotypes for these mutants using primers specific for each allele (specified on the left). PCR product lengths are specified on the right. <i>ACT1</i> was used as a positive control, and a no-DNA control was included (Control).</p

Strains used in this study.

<p>Strains used in this study.</p

Structure of the synthetic <i>cre</i> gene and Clox disruption cassettes.

<p>(A) Cartoon illustrating the components of the synthetic <i>cre</i> gene including the <i>CaMET3</i> promoter (<i>MET3_p:</i> grey), the two codon-optimized <i>cre</i> exons (blue), the <i>CaTUB2</i> intron, 3′-untranslated sequence from the <i>CaADH1</i> gene (yellow), the transcriptional terminator from <i>ScCYC1</i> (red), and engineered restriction sites for cloning. The DNA sequence of the synthetic <i>cre</i> exons and <i>CaTUB2</i> intron is provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100390#pone.0100390.s001" target="_blank">Figure S1</a>. (B) The <i>Clox</i> kit. Cartoons illustrating the structures of the <i>URA3-Clox</i> and <i>NAT1-Clox</i> cassettes (this study) and the <i>LUL, LHL</i> and <i>LAL</i> cassettes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100390#pone.0100390-Dennison1" target="_blank">[25]</a>; Black arrows, <i>loxP</i> sites; blue arrows, <i>MET3_p-cre</i> transcriptional start sites; open wavey boxes, common PCR priming sites for the disruption cassettes.</p

Cartoons illustrating the exploitation of <i>Clox</i> cassettes for multi-marker gene disruption and single marker recycling.

<p>(see text).</p

Validation of <i>URA3-Clox</i> for single marker recycling.

<p><i>URA3-Clox</i> was used to generate a homozygous <i>gsh2Δ/gsh2Δ</i> mutation in <i>C. albicans</i> RM1000 via single marker recycling (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100390#pone-0100390-g002" target="_blank">Figure 2</a>). (A) Confirmation of the expected phenotypes for wild type (<i>GSH2/GSH2</i>), unresolved heterozygous (<i>GSH2/gsh2Δ::URA3-Clox</i>), resolved heterozygous (<i>GSH2/gsh2Δ::loxP</i>), unresolved homozygous (<i>gsh2Δ::loxP/gsh2Δ::URA3-Clox</i>) and resolved homozygous mutants (<i>gsh2Δ::loxP/gsh2Δ::loxP</i>). Growth media are specified on the right: w/o, without a specific supplement; *, it was not possible to test the oxidative stress sensitivity of the unresolved <i>GSH2/gsh2Δ::URA3-Clox</i> and <i>gsh2Δ::loxP/gsh2Δ::URA3-Clox</i> strains because methionine and cysteine interfere with the oxidizing agent, t-BOOH. (B) PCR confirmation of the genotypes for these mutants using primers specific for each allele (specified on the left). PCR product lengths are given on the right. <i>ACT1</i> was used as a positive control, and a no-DNA control was included (Control).</p

High affinity copper acquisition influences fungal pathogenicity factors.

<p><i>C</i>. <i>albicans ctr1</i> cells are susceptible to copper deprivation imposed using the copper chelator BCS (95 μM) (A). The <i>C</i>. <i>albicans ctr1</i> mutant is also sensitive to iron chelation by BPS (B). The <i>C</i>. <i>albicans ctr1</i> strain is highly sensitive to H₂O₂ (C). Both of these defects are reversed by copper supplementation. Further, the <i>C</i>. <i>albicans ctr1</i> mutant displays growth defects on various carbon sources, a phenotype that is suppressed by copper supplementation, but not by haem or Fe³⁺ addition (D). Images were taken after 72 h incubation at 30°C. Number of cells spotted in every subset is given at the bottom of the panel. (E) The transcript levels for key metabolic genes are perturbed in <i>ctr1</i> cells, in the absence (top) and presence (bottom) of copper. These data represent averages of three biological replicates with two technical replicates. Numerical data are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158683#pone.0158683.s014" target="_blank">S10 Table</a>. (F) Changes in <i>C</i>. <i>albicans</i> transcript levels during renal colonisation. These data represent duplicate measurements from at least four biological replicates from the kidneys of animals at early (24 h, replicates E1–E4, 4 animals) or late (96 h, replicates L1–L5, 5 animals) infection stage. Colour scale is as in (E). Fungal transcript abundances were normalised to the <i>ACT1</i> mRNA. Numerical data are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158683#pone.0158683.s015" target="_blank">S11 Table</a>.</p

The <i>C</i>. <i>albicans CRP1</i> copper exporting ATPase is required for copper tolerance <i>in vitro</i> and plays a role in fungus-host interaction <i>in vivo</i>.

<p>(A) The deletion of <i>CRP1</i> severely impairs the ability of <i>C</i>. <i>albicans</i> to grow in the presence of copper. <i>C</i>. <i>albicans</i> cells were grown at 30°C overnight in YPD medium. Cultures were diluted at OD600 = 0.001 into fresh medium containing the appropriate CuSO₄ concentrations, and growth was monitored after 21 h, at 30°C. The values represent differences between the final and initial OD600 of cultures (+/- SD from two technical replicates), and are representative of three separate experiments performed. (B) Nevertheless, <i>CRP1</i> deletion does not impair the interaction of <i>C</i>. <i>albicans</i> with murine renal epithelial cells <i>in vitro</i>. The <i>CRP1</i> mutant elicits a similar degree of damage to the renal epithelial cell monolayer as the parental strain SC5314, as assessed by measurements of LDH release. At least three independent <i>C</i>. <i>albicans</i> inocula were used in at least two independent co-incubation experiments, in duplicate. (C) Similarly, there is no difference in the release of KC by the renal cells when incubated with the <i>crp1</i> mutant versus the SC5314 isogenic control.</p

Systemic candidiasis perturbs copper homeostatic functions in the liver and spleen.

<p>Progressive <i>C</i>. <i>albicans</i> infection affects the expression of copper homeostatic functions in the liver at transcript (A) and protein (B) levels. As the infection progresses, the abundance of hepatic transcripts for the metal storage protein metallothionein MT1 and the copper-containing ferroxidase ceruloplasmin (CP) increase, while the abundance of the copper importer <i>Ctr1</i> transcript decreases (A). In contrast, hepatic ceruloplasmin protein levels do not increase with progressive <i>C</i>. <i>albicans</i> infection, while CTR1 protein levels diminish (B), as assessed by immunohistochemistry (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158683#sec002" target="_blank">Materials and Methods</a>). All transcript data were acquired in duplicate from four biological replicates: healthy mice (replicates H1–H4), animals at early (24 h, replicates E1–E4) or late (96 h, replicates L1–L4) infection stages, following injection with saline (controls, H) or <i>C</i>. <i>albicans</i> SC5314. Transcript abundances were normalised against the <i>GAPDH</i> mRNA. Fold differences in expression are given when <i>p</i>≤0.05. Numerical data are provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158683#pone.0158683.s008" target="_blank">S4 Table</a>. (C) In the spleen, the red pulp macrophage population remains relatively constant, as revealed by detection of the red pulp macrophage antigen F4/80. The haem oxygenase HO-1, CTR1 importer and trans-Golgi ATPase ATP7B all decrease in the red pulp over the course of infection. Images are representative of four biological replicates. Brown colour indicates positive reaction and blue colour no reaction. Size bars: 5 mm (B) or 200 μm (C).</p