Fig 1.
Phylogenetic analysis of the predicted amino acid sequence of Vps45 or VpsB in selected fungi and humans by the neighbor-joining method.
A phylogenetic tree for Vps45 or VpsB was constructed with orthologous sequences retrieved from NCBI. An unrooted tree was created using the neighbor-joining consensus trees based on the calculated distances using 1,000 bootstrap replications. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site.
Fig 2.
Analysis of endocytosis by FM4-64 internalization.
A, B) Uptake of FM4-64 to assess endocytosis in WT, vps45 mutant, complemented (A), and GFP-tagged (B) strains grown for 24h in YNB-BPS at 30°C. Briefly, 1 x 106 cells/mL were inoculated in YNB-BPS + 100μM FeCl3, stained with 5 μM FM4-64 and transferred in a chamber slide where the cells were maintained at 30°C. Confocal microscopy images were taken every 15 min for 90 min. Intracellular acidification was assessed by flow cytometry on WT, vps45 mutants and complemented strains grown in YNB and YNB+150μM BPS at 30°C (C) and 37°C (D) and stained with carboxy-DCFDA. The differences in mean fluorescence intensity (ΔMFI) between stained and unstained cells are shown and reported in arbitrary units (A.U.). E) Growth of the WT, mutant and complemented strains was assessed in the presence of trafficking and glycosylation inhibitors. Cells were pre-cultured in YPD overnight at 30°C, serial diluted, and 5μL were spotted onto YPD plates containing 500nM N-Ethylmaleimide (NEM), 20μg mL-1 brefeldin A (BFA), 500μg mL-1 monensin, or 150ng mL-1 tunicamycin (TNC). Plates were incubated at 30°C or 37°C for 2 days.
Fig 3.
Growth of the WT, vps45 mutants and complemented strains in inorganic and host-related iron sources.
A) and B) Growth assays in liquid media with strains pre-grown in YNB+150μM BPS for 48h. After pre-growth, 1X105 of iron-starved cells/mL were inoculated in iron limited conditions supplemented with inorganic iron (i.e. YNB, YNB + 150μM BPS, YNB-BPS + 10μM FeCl3 (30°C) and + 25μM FeCl3 (37°C), YNB-BPS + 100μM FeCl3) and organic iron (i.e. YNB-BPS + 10μM Heme, + 2μg mL-1 Hemoglobin, + 50μg mL-1 Transferrin, + 0.05% sheep blood). The optical density (600nm) was recorded over a period of 5 days. C) Spot assays are shown for serial diluted WT, mutants and complemented strains in the same conditions as in liquid assays. Plates were incubated at 30°C and 37°C for 2–10 days. D) Inductively coupled plasma mass spectrometry (ICP-MS) measurements of iron content of pre-grown iron starved cells inoculated for 48h in YNB + 150μM BPS w/o 100μM FeCl3 at 30°C and 37°C.
Fig 4.
Localization of Cfo1-GFP in the presence and absence of Vps45.
A) Localization of Cfo1-GFP in low iron medium. Strains (WT, vps45Δ and complement) containing a CFO1-GFP construct were cultured overnight in YPD, washed 3 times and counted. Subsequently, 1X106 cells/mL were inoculated in YNB-BPS and incubated at 30°C for 1h. Then cells were stained with 5 μM FM4-64, transferred in a chamber slide and maintained at 30°C. Confocal images were taken every 15 min for 2h. B) Localization of Cfo1-GFP after 24h incubation in YNB, YNB + 150μM BPS, and YNB-BPS + 100μM FeCl3 at 30°C. The GFP label indicates evaluation of the green fluorescent protein and DIC indicates differential interference contrast microscopy.
Fig 5.
Impact of Vps45 on mitochondrial function.
A) and B) The association of Vps45-GFP with mitochondria was examined with the vps45 mutant complemented with VPS45-GFP. The strain was cultured in YNB and YNB + 150μM BPS for 24h at 30°C (A) and 37°C (B), cells were stained with 500nM MitoTracker Red CMXRos (mitotracker) and images were taken with a confocal microscope. C) The involvement of Vps45 in mitochondrial function and ROS sensitivity was examined by spot assays on inhibitors and H2O2. The WT, mutants and complemented strains were pre-cultured in YPD overnight at 30°C, serial diluted, and 5μL were spotted onto YPD plates containing either 5mM salicylhydroxamic acid (SHAM), 3μg mL-1 antimycin A, 10mM potassium cyanide (KCN) or 0.01% H2O2. Plates were incubated at 30°C and 37°C for 2 days. D) Mitochondrial membrane potential was assessed by flow cytometry on 50,000 cells stained with JC-1 (5μM) and grown in iron deplete and replete conditions at 30°C (D) and 37°C (E). Experiments were carried out at least two times in triplicate (ANOVA ** P < 0.001).
Fig 6.
Vps45 is involved in calcium homeostasis.
A) To examine the calcium-related phenotypes for the vps45 mutants, the WT, mutants and complemented strains were pre-cultured in YPD overnight at 30°C, serial diluted, and 5μL were spotted onto YPD plates containing either 25μM aminodarone, 100μg mL-1 cyclosporine A (CSA), 500ng mL-1 tacrolimus (FK506), or 5mM EGTA. Plates were incubated at 30°C and 37°C for 2 days. B) Growth curves of the WT, mutant and complemented strains inoculated in calcium-chelated media. Strains were pre-grown in YNB-EGTA for 48h and 1X105 of calcium-starved cells/mL were inoculated in YNB-EGTA, YNB-EGTA + 50mM calcium and incubated at (30°C) and (37°C). Optical density (600nm) was recorded over a period of 5 days. Disruption of VPS45 influences mitochondria morphology in calcium chelated conditions. Confocal images of mitochondria stained with 500nM mitotracker were taken of the WT, mutant and complemented strains inoculated for 24h in YNB ± 50mM EGTA at 30°C (C) and 37°C (D). Vps45 associates with mitochondria in calcium chelated conditions. The vps45 mutant complemented with VPS45-GFP was cultured in YNB + 50mM EGTA, for 24h at 30°C (E) and 37°C (F) and stained with mitotracker. Mitochondrial membrane potential was assessed by flow cytometry on 50,000 cells stained with JC-1 (5μM) and grown in calcium deplete conditions at 30°C and 37°C (G). Experiments were carried out at least two times in triplicate (ANOVA ** P < 0.001). H) Inductively coupled plasma mass spectrometry (ICP-MS) measurements of the calcium content of pre-grown iron starved cells inoculated for 48h in YNB + 150 μM BPS w/o 100μM FeCl3 at 30°C or 37°C. The same cells were used for iron measurements by ICP-MS in Fig 3D.
Fig 7.
Cell wall integrity is altered in vps45 mutants.
A-B) Spot assays of serial diluted WT, vps45 and crz1 mutants and vps45 complemented strains grown in the presence of cell wall stressors. Cells were pre-cultured in YPD overnight at 30°C, serial diluted, and 5μL were spotted onto YPD plates containing either 1.0 mg mL-1 calcofluor white (CFW), 0.01% sodium dodecyl sulfate (SDS), 0.5 mg mL-1 caffeine or 1.5M sodium chloride (NaCl). Plates were incubated at 30°C or 37°C and photographed after 48h of incubation. C) Flow cytometry measurement of differential fluorescence of cells stained with 100μg mL-1 pontamine, 100μg mL-1 concanavalin A, or 250μg mL-1 Eosin Y. Cells were grown in YPD at 30°C and 37°C for 24h and differences in the mean fluorescence intensity (ΔMFI) were determined between stained and unstained samples and reported in arbitrary units (A.U.). Analyses were performed on the gated WT unstained population. Capsule thickness, cell diameter measurements and GXM detection in supernatants (D-G) of cell grown in defined limited iron media (33) at 30°C (D) and 37°C (F). Cells were grown in LIM at 30°C for 48 h and the capsule thickness and cell diameter of at least 40 cells of each strain were measured. Each bar represents the average with standard deviations. Statistical significance relative to WT is indicated by an asterix (ANOVA ** P < 0.0001). Shed polysaccharides were collected from culture supernatants of all strains incubated at 30°C (E) or 37°C (G). The electrophoretic mobility and relative quantities of shed GXM were assessed by immunoblotting with the anti-GXM antibody mAb18b7.
Fig 8.
Vps45 is involved in resistance to drugs that target the membrane and vacuole.
The WT, mutants and complemented strains were grown in the presence of antimalarial and antifungal drugs. Cells were pre-cultured in YPD overnight at 30°C, serial diluted, and 5μL were spotted onto YPD plates containing either 10 μg mL-1 caspofungin, 1.6 mM quinacrine, 6 mM chloroquine, 10 μg mL-1 fluconazole and 0.05 μg mL-1 miconazole (A). Growth with supplementation of 100 μM feCl3 and 100 μM heme to antimalarial and antifungal drugs was assessed (B). Plates were incubated at 30°C or 37°C for 2 days.
Fig 9.
Vps45 is required for survival in macrophages and virulence in mice.
A) WT, vps45 mutants and complemented strains were incubated with the murine macrophage-like cell line J774A.1. The strains were incubated for 2 h and 24 h in DMEM with macrophages at a MOI of 1:1 at 37°C. CFUs from lysed macrophages were obtained and survival was calculated as the ratio of CFUs at 24 h versus 2 h of incubation. The data represent the mean values ± standard error of the mean of four independent biological experiments done in triplicate. Statistical analysis was performed using an unpaired two-tailed Student's t test using WT as the reference strain (*** P < 0.0001). B) 10 female BALB/c mice were inoculated intranasally with 2X105 fungal cells and survival of the mice was monitored daily. Survival differences between groups of mice were evaluated by log-rank test against WT. (*** P < 0.0001). C) Fungal burden was determined in systemic organs (lungs, brain, liver, kidney and spleen) and in cardiac blood for all mice at time of death. The Mann-Whitney U test was used for statistical analysis using WT as the reference strain (** P < 0.005, *** P < 0.0001).
Fig 10.
Schematic representation of the role of Vps45 in endocytosis of exogenous iron sources and in intracellular trafficking from the plasma membrane to the vacuole and mitochondria.
The internalization of newly created endosomes loaded with the high affinity iron uptake system (Cft1-Cfo1) and heme and a putative receptor (?) is depicted. These endosomes may fuse into a pre-vacuolar compartment also called a multivesicular body (MVB) and undergo progressive acidification of the intraluminal milieu until fusion with the vacuole. Multiple membrane fusion events occur during that process and endosomal cargo is ultimately delivered into the vacuole for dissociation and degradation. Direct fusion from iron-loaded endosome to the mitochondrial membrane is also possible [59], but has yet to be demonstrated in C. neoformans. An exchange of iron and calcium ions between the vacuole and mitochondria is likely and may require Vps45 for vCLAMP formation [37].