<i>ZRT1</i> and <i>PRA1</i> are required for microcolony development on endothelia in the absence of exogenous zinc.

<p>Single cells of <i>C. albicans</i> wild type (M1477), <i>zrt1</i>Δ (M2006) or <i>pra1</i>Δ (M2008) were incubated for 16 h in zinc-depleted cell culture medium on endothelial monolayers (Endothelium) or on plastic (Ctrl). Experiment was performed three times. representative images are shown. Note that only wild type cells were able to assimilate sufficient zinc for microcolony development.</p

<i>In silico</i> prediction of Pra1-zinc binding.

<p>(<b>A</b>) Primary amino acid sequence of Pra1 (<i>Candida</i> Genome Database) with zinc-binding motifs in red. (<b>B</b>) Three-dimensional model of Pra1 built with Phyre². (<b>C</b>) and (<b>D</b>) close-up of predicted zinc coordination sites. Note the presence of arginine, rather than glutamic acid at residue 179 (<b>C</b>) and that the C-terminal tail of Pra1 has multiple additional potential zinc binding histidine residues (<b>D</b>).</p

Invasive <i>C. albicans</i> hyphae sequester endothelial zinc.

<p>(<b>A</b>) Endothelial monolayers were infected with wild type <i>C. albicans</i> (M134) for 3.5 h in zinc-free medium. As a control (Ctrl), <i>C. albicans</i> was incubated under identical conditions in the absence of endothelium. Concanavalin A (ConA) stains the entire fungus red; anti-<i>Candida</i> antibody (Anti-Ca-Ab) stains only the extracellular (non-invasive) part of the fungus green; zinquin stains zinc blue. Note that the invasive (ConA⁺/Anti-Ca-Ab⁻) sections of <i>C. albicans</i> hyphae stain positive for zinquin. (<b>B</b>) Quantification of hyphal length of <i>C. albicans</i> incubated for 3.5 h in the absence or presence of endothelia, either with or without zinc supplementation. (<b>C</b>) Quantification of zinquin intensity of <i>C. albicans</i> hyphae incubated in zinc-free medium in the absence (Ctrl) or presence of Endothelium; additionally, zinquin intensity of intracellular (invasive) and extracellular (non invasive) hyphae was determined. Experiment was performed twice in triplicate. Asterisks indicate significance (<i>P</i><0.001) by Student's t-test.</p

Deletion of <i>PRA1</i> precludes host zinc sequestration.

<p>(<b>A</b>) Endothelial monolayers were infected with wild type (M134), <i>pra1</i>Δ (M1809) or <i>pra1</i>Δ+<i>PRA1</i> (M1785) <i>C. albicans</i> strains for 3.5 h in zinc-free medium. Concanavalin A (ConA) stains the entire fungus red; anti-<i>Candida</i> antibody (Anti-Ca-Ab) stains only the extracellular (non-invasive) part of the fungus green; zinquin stains zinc blue. Note that <i>pra1</i>Δ does not accumulate zinc from the host cell. (<b>B</b>) Quantification of hyphal length of <i>C. albicans</i> in association with endothelia in either zinc free medium (−Zn) or with supplementation with 20 µM zinc (+Zn). (<b>C</b>) Quantification of zinquin fluorescence intensity per area of the invasive (intracellular) portion of <i>C. albicans</i> hyphae. Experiment was performed twice in triplicate. Asterisks indicate significance (<i>P</i><0.001) by Student's t-test.</p

Synteny of genes encoding zinc transporters and zinc-binding proteins.

<p>Genomic arrangement of <i>C. albicans ZRT1</i> and <i>PRA1</i> orthologues in selected fungal species. Only direct orthologues of <i>C. albicans ZRT1</i> are shown (CaZrt1-cluster, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002777#ppat.1002777.s005" target="_blank">Figure S5</a>). Note that synteny is conserved between distantly related species (e.g. <i>C. albicans</i> and <i>U. maydis</i>) but has broken down in more closely related species (e.g. <i>C. albicans</i> and <i>C. parapsilosis</i>).</p

Model of <i>C. albicans</i> zinc scavenging from host cells.

<p>Following host cell invasion, Pra1 is expressed and secreted. The released fraction of Pra1 binds zinc, either directly from a cellular pool or from host zinc-binding proteins. Reassociation with the cell surface of <i>C. albicans</i> is mediated via direct Pra1-Zrt1 interaction.</p

Endothelial damage is zinc-dependent in the absence of <i>PRA1</i>.

<p>Endothelial monolayers were infected with wild type (M134), <i>pra1</i>Δ (M1809) or <i>pra1</i>Δ+<i>PRA1</i> (M1785) <i>C. albicans</i> in zinc-free medium with indicated zinc-supplementation. Following 24 h co-incubation, endothelial damage was assessed by measuring the release of lactate dehydrogenase. Experiment was performed 3 times in triplicate. Asterisks indicate significance by Student's t-test: * <0.05; ** <0.01; *** <0.001.</p

Pra1 is a zinc binding protein.

<p>(<b>A</b>) Recombinant Pra1 or β-galactosidase were pre-incubated with zinc, loaded on 10 kDa microspin columns and sequentially washed with Hs buffer. The zinc content of each flow-through was determined by PAR assay. (<b>B</b>) The fully washed proteins were proteolytically digested and released zinc measured by PAR assays. Experiment was performed 3 times in duplicate.</p

Additional file 1: Figure S1. of Age-related macular degeneration associated polymorphism rs10490924 in ARMS2 results in deficiency of a complement activator

Expression vector with ARMS2 coding sequence. (PDF 268 kb