Zrc1 is required for virulence in a <i>Galleria</i> infection model.

<p><i>Galleria</i> larvae (10 per group) were infected with 10⁵ <i>C</i>. <i>albicans</i> cells and monitored every 12 h. Note that whilst wild type result in high mortality, only one <i>zrc1</i>Δ-infected larvae died. Experiment performed twice—here, and in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007013#ppat.1007013.s009" target="_blank">S8 Fig</a>. <i>zrc1</i>Δ is significantly attenuated compared to wild type (P = 0.0001) and <i>zrc1</i>Δ+<i>ZRC1</i> (P = 0.0009), but not compared to PBS control (P = 0.3173); Log-rank (Mantel-Cox) test.</p

Relationship between Zrc1, zincosomes and zinc tolerance.

<p>(A) Cells were challenged with potentially toxic zinc (1 mM), stained with zinquin and fluorescence determined. <i>P</i> < 0.0001 compared to wild type and revertant. (B) Micrographs of cells treated as in A. Note that <i>zrc1</i>Δ is highly defective for zincosome formation in response to 1 mM ZnSO₄ –a condition under which wild type, but not <i>zrc1</i>Δ cells can grow (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007013#ppat.1007013.s008" target="_blank">S7 Fig</a>).</p

pH-dependent functionality and regulation of Zrt1 and Zrt2 in <i>C</i>. <i>albicans</i>.

<p>(<b>A</b>) Zrt2 is essential in acidic medium. Indicated strains, precultured in YPD, were washed and cultured in SD (YNB+glucose) medium alone, or supplemented with 100 μM ZnSO4 or with 50 mM HEPES pH 7.4. Asterisks indicate statistical significance compared to the wild type; # indicates statistical significance compare to the <i>zrt2</i>Δ in SD; P <0.05. (<b>B</b>) <i>ZRT1</i> promoter activity is pH regulated and <i>ZRT2</i> is constitutively expressed under zinc limitation. (<i>P</i>_<i>ZRT1</i>-GFP and <i>P</i>_<i>ZRT2</i>-GFP reporter strains in LZM buffered to indicated pH values). LZM was used due to lower green autofluorescence. Experiment performed three times. (<b>C</b>) Double deletion of <i>ZRT1</i> and <i>ZRT2</i> precludes growth at both acidic and neutral alkaline pH. Strains were cultured as in (A) and growth kinetics measured over 48 h in a microtitre plate. Experiment performed twice in triplicate.</p

Zincosome formation is Zrc1 dependent.

<p>(A) Zincosome screen. Wild type, ZnT deletion mutants and <i>zrc1</i>Δ+<i>ZRC1</i> strains were pulsed with 25 μM zinc for 20 minutes and zincosome fluorescence determined by staining with zinquin. Prepulsed cells were also stained as control. Experiment was performed at least twice in duplicates and all data normalised to the post-pulse value of wild type. ANOVA was first performed on initial (pre-normalised data). Asterisks indicate statistical significance compared to wild type and to relevant deletion mutant ** P <0.01. (B) As panel A, except zinquin fluorescence kinetics was determined by flow cytometry. Experiment performed three times. <i>zrc1</i>Δ exhibits significantly reduced zinquin fluorescence compared to wild type and revertant at 20 minutes <i>P</i> < 0.001, ANOVA.</p

Zrc1 is essential for zinc detoxification.

<p>(<b>A</b>) Strains were cultured for 24 h in SD0 medium containing indicated zinc supplementation. Experiment performed at least three times in duplicate for zinc concentrations at 25 μM and above. *** indicates significant difference (<i>P</i> < 0.001) compared to wild type and revertant, ANOVA. (<b>B</b>) Strains were precultured in SD0, challenged with 1 M ZnSO₄ for 3 h and viability assessed by measuring CFUs. ** indicates significant difference (<i>P</i> < 0.01) compared to wild type and revertant, ANOVA. Experiment performed three times for wild type and <i>zrc1</i>Δ and twice in duplicate for all three strains.</p

<i>C</i>. <i>albicans</i> Zrt2 is required for kidney colonisation in the presence of functional calprotectin.

<p>Indicated mice strains were infected with indicated fungal strains and kidney colonisation determined by plating CFUs on day one and day three post-infection. At day three post-infection, <i>C</i>. <i>albicans</i> wild type kidney fungal burden had increased significantly by 6.5-fold (<i>P</i> = 0.034), Deletion of <i>ZRT2</i> precluded an increase in kidney fungal burden between day one and day three post-infection (<i>P</i> = 0.597), asterisk. Complementation of <i>zrt2</i>Δ with a single copy of <i>ZRT2</i> restored kidney colonisation at day three (4.5-fold higher than at day one, <i>P</i> = 0.004).</p

Identified ZnT-type transporter in <i>C</i>. <i>albicans</i> and their relationship with <i>S</i>. <i>cerevisiae</i>.

<p>Identified ZnT-type transporter in <i>C</i>. <i>albicans</i> and their relationship with <i>S</i>. <i>cerevisiae</i>.</p

<i>P</i>_<i>ZRT1</i> and <i>P</i>_<i>ZRT2</i> metallo-regulation is zinc specific.

<p>Excess (100 μM) zinc, but not iron, manganese or copper downregulate <i>P</i>_<i>ZRT1</i>-GFP (<b>A</b> and <b>B</b>) and <i>P</i>_<i>ZRT2</i>-GFP (<b>C</b> and <b>D</b>). Experiment was performed three times. *(P <0.05) and *** (P <0.0001) = significantly different from LZM, Student’s t-test.</p

Zrt2 protects against calprotectin-dependent inhibition of fungal growth during <i>C</i>. <i>albicans</i>-neutrophil extracellular trap interaction.

<p>Indicated strains were incubated with wild type or S100A9-/- -derived NETs or in medium only. Following ~21 hours incubation, metabolic activity was determined by XTT assay. Activity in the presence of both NET groups was determined compared to control conditions in the absence of NETs. Experiment was performed three time. * indicates P < 0.05 and # not significantly different to wild type, Students t-test Data presented are fold reduction in activity due to the presence of calprotectin.</p

Growth of <i>zrt2</i>Δ strains is specifically rescued by excess zinc.

<p>Indicated strains were cultured as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007013#ppat.1007013.g001" target="_blank">Fig 1</a> with zinc, iron, manganese (100 μM) or copper (10 μM) and growth kinetics measured over 36 h in a microtitre plate. Experiment performed twice in triplicate. Iron had a moderate inhibitory effect on <i>C</i>. <i>albicans</i> growth. Note that only zinc rescued growth of <i>zrt2</i>Δ strains.</p