Fig 1.
C. albicans GDH2 is required for growth using amino acids as sole carbon and nitrogen sources.
(A) Schematic diagram of arginine/proline catabolism. Arginine is catabolized to proline in the cytoplasm, proline is transported into mitochondria, proline is catabolized to glutamate in two enzymatic reactions, catalyzed by FAD-dependent proline oxidase (PUT1) and NAD+-linked Δ1-pyrroline-5-carboxylate (P5C) dehydrogenase (PUT2), respectively. The two central reactions for nitrogen source utilization are catalyzed by NADH-dependent glutamate synthase (GLT1) and NAD+-linked glutamate dehydrogenase (GDH2). The gene products and metabolic steps marked in red are localized to the mitochondria. (B) YPD grown put2-/- (CFG318), put1-/- (CFG154), put1-/- put2-/- (CFG159), dur1,2-/- (CFG246), wildtype (WT, SC5314) and gdh2-/- (CFG279) cells were washed, resuspended at an OD600 ≈ 0.05 in YNB+CAA containing the pH indicator bromocresol purple, and the cultures were incubated shaking at 37°C for 16 h. (C) Wildtype (WT, SC5314), gdh2-/- (CFG279), dur1,2-/- (CFG246) and CRISPR control (CFG182) cells were pre-grown in YPD, washed, resuspended at an OD600 ≈ 1, and 5 μl were spotted onto the surface of solid YNB + CAA with bromocresol purple without and with the indicated carbon source. The plates were incubated at 37°C for 72 h. The images are representative of at least 3 independent experiments. (D) Volatile ammonia released from strains as in (C); the results are the average of at least 3 independent experiments (Ave. ± CI; **** p ≤ 0.0001).
Fig 2.
C. albicans Gdh2 localizes to the mitochondria and environmental alkalization requires mitochondrial function.
(A) Gdh2-GFP co-localizes with the mitochondrial marker MitoTracker Deep Red FM (MTR). YPD grown cells expressing GDH2-GFP (CFG273) were harvested, washed, grown in SED (0.2% glucose) or YNB + CAA at 37°C for 24 h, and stained with 200 nM MTR prior to imaging by differential interference contrast (DIC) and confocal fluorescence microscopy; the scale bar = 5 μm. (B) Wildtype cells (SC5314) from overnight YPD cultures were washed and then diluted to either OD600 ≈ 0.1 (top panel) or ≈ 5 (bottom panel) in liquid YNB+CAA with the indicated concentrations of mitochondrial complex III inhibitor antimycin A. Cultures were grown at 37°C under constant aeration for 24 h and 2.5 h, respectively, and photographed. To assess viability after Antimycin A treatment, inhibited cells from 24 h old culture (top panel) were harvested, washed, and then resuspended in fresh YNB+CAA media and incubated for 24 h (up to 48 h) at 37°C (middle panel). Images are representative of at least 3 independent experiments.
Fig 3.
GDH2 expression is repressed by glucose.
(A) Live cell imaging of Gdh2-GFP expression in cells (CFG273) shifted from YPD to YNB+CAA. Cells were pre-grown in YPD, transferred to a thin agar slab of YNB+CAA medium and observed by confocal microscopy at 37°C. Growth and the intensity of mitochondrial-localized GFP signal were monitored every hour for 6 h (images at 2 h intervals are shown). A region in the 6 h micrograph is enlarged (see inset). (B) Gdh2-GFP expression is rapidly induced in cells shifted from YPD to YNB+CAA. Cells were pre-grown in YPD and used to inoculate liquid YNB+CAA (OD600 ≈ 2.0); at the times indicated, the pH was measured (right panel; average of 3 independent experiments) and the levels of Gdh2-GFP expression (left panel) were monitored by immunoblot analysis. (C) Gdh2 expression is carbon source dependent. (upper panels) Cells grown in YPD (OD600 ≈ 2.0) were harvested, transferred to YPG (YP + 1% glycerol; lanes 1–3) and after subsampling at 6 h, 2% glucose was added to cultures (lanes 5–6); YPD grown cells (OD600 ≈ 2.0) were shifted to YNB + CAA with 2% glucose (lanes 7–10), 0.2% glucose (lanes 11–14) or 1% glycerol (lanes 15–18) and grown at 37°C. Extracts were prepared at the times indicated and the levels of Gdh2-GFP and tubulin (loading control) were assessed by immunoblotting using primary α-GFP and α-tubulin antibodies. (Lower panel) Photograph of tubes after 6 h of growth in YNB+CAA containing BCP without an additional carbon source (No addition), supplemented with 2% glucose, 0.2% glucose or 1% glycerol as indicated.
Fig 4.
Gdh2 is dispensable for filamentous growth on solid media and is induced in C. albicans cells phagocytized by murine macrophages.
(A) Wildtype (WT, SC5314), gdh2-/- (CFG279), and CRISPR control (CFG182) strains, pre-grown in YPD, were washed, resuspended at an OD600 ≈ 1 in water, and 5 μl aliquots were spotted on solid Spider and Lee’s media. Representative colonies were photographed 5 days after incubation at 37°C. CFG273 cells were co-cultured in CO2-independent medium with (B) RAW264.7 macrophages, pre-stained with Lysotracker Red (LST) at MOI of 1:1 (C:M). The co-cultures were followed by live cell imaging. Micrographs were taken at the times indicated (S1 Vid). In each series, three CFG273 cells are marked prior to (open arrows) and after (closed arrows) being phagocytized. (C) Gdh2 expression in phagocytized fungal cells (CFG324) expressing Gdh2-GFP and PADH1-RFP). RAW264.7 macrophages were co-cultured with opsonized CFG324 for 1 h in HBSS prior to measuring GFP and RFP intensities in non- (ext) and phagocytized (int) fungal cells. Results from 3 biological replicates (≥ 100 ext and int cells/replicate) are shown (Ave. ± CI; **** p ≤ 0.0001 by Student t-test).
Fig 5.
Competition assay to compare wildtype and gdh2-/- filamentation and survival upon phagocytosis by primary BMDM.
(A) (left panels) Wildtype (WT; PADH1-GFP; SCADH1G4A) or gdh2-/- (PADH1-RFP, CFG275) cells was co-cultured with primary BMDM (MOI of 3:1; C:M) for 30 min in HBSS. Non-phagocytized fungal cells were removed by washing and the co-cultures were monitored by live cell imaging for 4 h (SV3 and SV4 Vids). (right panel) Candidacidal activity of BMDM. Untagged WT (PLC005) and gdh2-/- (CFG279) strains were co-cultured for 2 h with BMDM at MOI of 3:1 (without washing) and then CFUs recovered and compared to the CFUs in the starting inoculum. No significant difference in the co-culture survival between the strains using Student t-test; data presented are average of 4 biological replicates. (B) Competition assay. The same strains as in (A) were mixed 1:1, co-cultured for 30 min in HBSS, washed extensively to remove external cells, and the interactions were followed by TLM for 5 h (S5 Vid). Solid arrows indicate macrophages with phagosomes containing both WT and gdh2-/- cells; open arrows indicate macrophages with phagosomes containing either WT (green) or gdh2-/- (red) cells. The observed growth of WT and gdh2-/- cells within a single macrophage is schematically illustrated (right upper panel). For the WT to gdh2-/- ratio determination following co-culture, mutants were mixed 1:1 and then the mixed cells were used to infect BMDM at MOI of 3:1. After an initial macrophage lysing step, the ratio of WT:gdh2-/- was analyzed by plating serially diluted cells on YPD and then testing at least 50 independent colonies for their ability to alkalinize YNB+CAA medium (pH = 4). The ratio of recovered cells (after 2 h) was compared to the input ratio. No significant difference in the WT:gdh2-/- ratio between input and recovered using Student t-test; data were obtained from six (6) biological replicates.
Fig 6.
Phagosomal acidification following phagocytosis.
(A) Wildtype (PLC005) and gdh2-/- (CFG279) cells were labeled with pHrodo, and co-cultured with RAW macrophages in HBSS at MOI of 3:1 (C:M); images were obtained at 30 min intervals for 4 h. Representative images and quantification of phagosome intensities for both strains. (B) (upper panels) Time lapse images showing the pH changes encountered by a single fungal cell phagocytosed by a RAW macrophage. Time = 0 min is arbitrarily set when a single C. albicans (gdh2-/-; CFG279) cell is about to be phagocytized by the macrophage. Black arrow indicates the macrophage containing the fungal cell followed for quantification of fluorescence intensities (lower panel). The time points with maroon color correspond to the time images in the upper panels were taken. The asterisk (*) and bracket indicate a period of time with apparent rapid changes in fluorescence intensity, the consequence of phagosomes moving in and out of focus (S12 Vid).
Fig 7.
Virulence of wildtype and gdh2-/- C. albicans in Drosophila and murine systemic infection models.
(A) D. melanogaster BomΔ55C flies were infected with wildtype (SC5314) or gdh2-/- (CFG279) cells as indicated, and the survival of flies was followed for six days. Each curve represents the average of a minimum of three independent infection experiments (20 flies/strain) performed on different days. (B) Groups of C57BL/6 mice (n = 10) were infected via the lateral tail vein with 3x105 CFU of C. albicans wildtype or gdh2-/- cells (upper panels) and survival (left) and weight loss (right) was monitored at the timepoints indicated. Survival curves from two independent experiments were statistically analyzed by the Kaplan-Meier method (a log-rank test, GraphPad Prism), no significant difference. The fungal burden (lower panels) in brain (left), kidney (middle), and spleen (right) extracted from mice 3 days post infection. Each symbol represents a sample from an individual mouse and results were compared by Student t-test, no significant difference. (C) Competition assay; mice were infected via the tail vein with an inoculum (I) comprised of an equal number of wildtype (SC5314) and gdh2-/- (CFG279), 1:1. At 3 days post infection, the abundance and genotype of fungal cells recovered from kidneys was quantitated and the ratio of wildtype:gdh2-/- recovered (R) was determined. The significance of the log2(R/I) values was assessed using an unpaired Student t-test, no significant difference (ns).