Figure 1.
Invasive C. albicans hyphae sequester endothelial zinc.
(A) Endothelial monolayers were infected with wild type C. albicans (M134) for 3.5 h in zinc-free medium. As a control (Ctrl), C. albicans was incubated under identical conditions in the absence of endothelium. Concanavalin A (ConA) stains the entire fungus red; anti-Candida 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 C. albicans hyphae stain positive for zinquin. (B) Quantification of hyphal length of C. albicans incubated for 3.5 h in the absence or presence of endothelia, either with or without zinc supplementation. (C) Quantification of zinquin intensity of C. albicans 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 (P<0.001) by Student's t-test.
Figure 2.
In silico prediction of Pra1-zinc binding.
(A) Primary amino acid sequence of Pra1 (Candida Genome Database) with zinc-binding motifs in red. (B) Three-dimensional model of Pra1 built with Phyre2. (C) and (D) close-up of predicted zinc coordination sites. Note the presence of arginine, rather than glutamic acid at residue 179 (C) and that the C-terminal tail of Pra1 has multiple additional potential zinc binding histidine residues (D).
Figure 3.
Pra1 is a zinc binding protein.
(A) 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) The fully washed proteins were proteolytically digested and released zinc measured by PAR assays. Experiment was performed 3 times in duplicate.
Figure 4.
Deletion of PRA1 precludes host zinc sequestration.
(A) Endothelial monolayers were infected with wild type (M134), pra1Δ (M1809) or pra1Δ+PRA1 (M1785) C. albicans strains for 3.5 h in zinc-free medium. Concanavalin A (ConA) stains the entire fungus red; anti-Candida antibody (Anti-Ca-Ab) stains only the extracellular (non-invasive) part of the fungus green; zinquin stains zinc blue. Note that pra1Δ does not accumulate zinc from the host cell. (B) Quantification of hyphal length of C. albicans in association with endothelia in either zinc free medium (−Zn) or with supplementation with 20 µM zinc (+Zn). (C) Quantification of zinquin fluorescence intensity per area of the invasive (intracellular) portion of C. albicans hyphae. Experiment was performed twice in triplicate. Asterisks indicate significance (P<0.001) by Student's t-test.
Figure 5.
Endothelial damage is zinc-dependent in the absence of PRA1.
Endothelial monolayers were infected with wild type (M134), pra1Δ (M1809) or pra1Δ+PRA1 (M1785) C. albicans 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.
Figure 6.
PRA1 expression is zinc regulated.
C. albicans harbouring GFP under control of either the ACT1 (PACT1-GFP, M135) or PRA1 (PPRA1-GFP, M1522) promoter were incubated in Lee's medium, pH 7.4 either with or without supplementation with (100 µM) zinc. GFP fluorescence was determined at indicated time points and normalised against a C. albicans strain harbouring an empty vector. Note that the addition of zinc fully suppresses expression from the PRA1 promoter. Experiment was performed three times in triplicate.
Figure 7.
ZRT1 and PRA1 are required for microcolony development on endothelia in the absence of exogenous zinc.
Single cells of C. albicans wild type (M1477), zrt1Δ (M2006) or pra1Δ (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.
Figure 8.
Synteny of genes encoding zinc transporters and zinc-binding proteins.
Genomic arrangement of C. albicans ZRT1 and PRA1 orthologues in selected fungal species. Only direct orthologues of C. albicans ZRT1 are shown (CaZrt1-cluster, see Figure S5). Note that synteny is conserved between distantly related species (e.g. C. albicans and U. maydis) but has broken down in more closely related species (e.g. C. albicans and C. parapsilosis).
Figure 9.
Zrt1 is required for reassociation of soluble Pra1 to the fungal cell.
(A) Predicted three-dimensional structures and interaction of Pra1 and Zrt1. The large extracellular domain of Zrt1 represents the N-terminal tail from amino acids 1–183. (B) C. albicans wild type (M1477), zrt1Δ (M2006) and zrt1Δ+ZRT1 (M2010) were exposed to recombinant His-tagged Pra1 and binding visualised with a fluorescently conjugated anti-His antibody (experiment was performed twice in triplicate); (C) additionally, binding of rPra1 was assessed by Western Blot hybridisation (experiment was performed twice). Note that deletion of ZRT1 precludes reassociation of Pra1 with the fungal cell.
Figure 10.
Model of C. albicans zinc scavenging from host cells.
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 C. albicans is mediated via direct Pra1-Zrt1 interaction.