Fig 1.
High and low Cu stressed cuf1Δ cells show a decrease in cell wall chitin.
(A) Calcofluor white (CFW) staining of WT and cuf1Δ cells in high and low copper stress. Cells were cultivated at high copper stress (SC+ 100 μM CuSO4) or low copper stress (SC + 100 μM BCS) for 12h at 30°C and stained with CFW as a marker of total cell wall chitin/chitosan. (B) Quantification of CFW staining signal. Shown is the mean +/- SEM of the relative CFW intensity from 4 independent experiments. The CFW intensities were measured with ImageJ/Fiji and normalized to cell count. Shown is the relative CFW intensity (low copper WT set to 1). A 2-way ANOVA was performed from log transformed data using GraphPad Prism.
Fig 2.
The Cuf1 transcription factor as well as its targets Cbi1 and Ctr1 are involved in maintaining cell wall integrity during copper deficiency.
(A) Transcript abundance of the cell wall stress gene ROM2 during copper deprivation. The WT, cuf1Δ and Cuf1-Flag complemented cuf1ΔC strains were incubated in either high copper (SC medium with 1 mM CuSO4 for 1h at 30°C) or low copper (SC medium with 1 mM BCS for 6h at 30°C) conditions. Quantitative RT-PCR was used to assess relative ROM2 transcript levels for each strain compared to the WT in each condition. Presented is the mean +/- SEM of the relative transcript levels of 5 biological replicates. A 2-way ANOVA was performed from log transformed data using GraphPad Prism. (B) Growth analysis in presence of cell wall/ surface stressors. Five-fold serial dilutions of cell suspensions for each strain were incubated on SC medium with 100 μM BCS, supplemented with indicated amounts of cell wall and cell surface stressors. Plates were incubated at 30°C for 2-4d, and the figure shows a representative image from 3 independent experiments. (C) GAL7 promoter-driven expression of Cbi1 in different mutant strain backgrounds during cell wall stress. Five-fold serial dilutions of cell suspensions for each strain were incubated on SC medium with indicated copper concentrations, in the presence or absence of the cell wall stressor Congo red, on SC medium with either glucose (SC-Glu) (CBI1-repressing conditions) or galactose (SC-Gal) (CBI1-inducing conditions) as carbohydrate source. Plates were incubated at 30°C for 3d. This figure shows a representative image from 3 independent spotting experiments. (D) The WT, cbi1Δ, and cbi1Δ (cbi1ΔC-WT) complemented strains were incubated in YP medium with the non-fermentable carbon sources ethanol and glycerol (YPEG) in presence and absence of the cell wall stressor SDS, and with or without the addition of 5 μM CuSO4. Growth was assessed by OD600 of the cultures after 72h of incubation at 30°C and normalized to WT growth in YPEG media. Presented is the mean +/- SEM of the relative growth rates of 3 biological replicates. 1-way ANOVA was performed from log transformed data using GraphPad Prism. (E) Impact of the ccc2Δ mutation on growth in presence of the cell wall stressor Congo red. Five-fold serial dilutions of cell suspensions for each strain were incubated on SC medium with and without the addition of Congo red, and with 100 μM or 500 μM BCS as copper chelation. Plates were incubated at 30°C for 2-4d. (F) Copper restoration of cell wall stressor sensitivity of cbi1Δ cells. Five-fold serial dilutions of cell suspensions for each strain were incubated on SC medium with and without 100 μM BCS, and with the addition of either 100 μM CuSO4 or 100 μM FeCl3. Plates were incubated at 30°C for 3d. This figure shows a representative image from 3 independent spotting experiments.
Fig 3.
Copper deficient cbi1Δ cells show a distinct cell wall electron density pattern, which is not due to overall difference in cell associated copper levels or copper adsorption ability.
Transmission electron microscopy (TEM) images of copper sufficient or deficient WT and cbi1Δ cells. The WT and cbi1Δ strains were incubated in YPD medium supplemented with 10 μM CuSO4 (Cu sufficiency) or with 250 μM BCS (Cu deficiency) for 24h at 30°C. (A) Representative images with representative cell wall thickness indicated (29000 x magnification). (B) Quantification of cell wall thickness. Measurements were performed using the ImageJ/Fiji measurement tool: Cu sufficiency-WT 10 cells, Cu sufficiency- cbi1Δ 8 cells, Cu deficiency-WT 11 cells and Cu deficiency-cbi1Δ 14 cells. A 1-way ANOVA was performed using GraphPad Prism from log transformed data. (C) Quantification of the cell wall staining intensity. Presented is the analysis of two sets of TEM images. The gray value was measured with ImageJ/Fiji and plotted against the distance along the cell wall from the inner cell surface. (D-E) ICP-MS based metal quantification of cell associated copper (D) and Iron (E) in pg metal per 106 cells. Indicated strains were grown in identical growth conditions to the TEM experiment. Presented is the average +/- SEM from 3 biological replicates. A 2-way ANOVA was performed from log transformed data using GraphPad Prism. (F) ASV analysis of copper adsorption by Cn cells. Indicated strains were grown under Cu starvation (YPD + 250 μM BCS) for 24h at 30°C. Copper was titrated in 250 nM steps into 20 mL cell suspension of indicated concentration or PBS (= control). The peak height of the resulting current in each titration step was measured using the NOVA software (Autolab) and plotted against copper concentration. Linear regression was performed to determine slope of each titration experiment. Shown is the mean +/- SEM of the slopes determined by 3 independednt titration experiments. A ONE-Way ANOVA was performed from log transformed data.
Fig 4.
Cbi1-dependent cell wall gene transcript abundance and cell wall chitosan levels.
(A)-(B) The WT, cbi1Δ mutant, and cbi1ΔC-WT complemented strains were incubated in YPD supplemented with 250μM BCS for 24h at 30°C. Relative transcript abundance for cell wall genes involved in (A) chitin/chitosan synthesis or (B) glucan synthesis was assessed by quantitative RT-PCR and normalized to WT. Presented data is the mean +/- SEM of the relative transcript levels of 4 biological replicates. A 1-way ANOVA was performed from log transformed data using GraphPad Prism. (C)-(D) Chitin/chitosan quantification of copper sufficient (C) or deficient (D). The WT, cbi1Δ mutant, and cbi1ΔC-WT complemenbted strains were incubated for 24h in YPD+ 10 uM CuSO4 (copper sufficiency) or YPD +250 uM BCS (copper deficiency). MBTH-based chitin/chitosan quantification (μM glucosamin/107 cells) was performed for purified cell wall material. Data represent the mean +/- SEM of 3 biological replicates. A 1-way ANOVA was performed from log transformed data using GraphPad Prism. (E) The same strains incubated in identical conditions above were also assessed for chitin and chitosan by staining with calcofluor white (CFW) (total chitin/chitosan), wheat germ agglutin (WGA)-Alexa 488 (exposed chitin/chitosan), and (F) Eosin Y (chitosan). Representative images from at least three independent experiments are demonstrated.
Fig 5.
Cbi1 depletion effects chitin architecture during copper deficiency and host stress.
(A)-(D) The WT, cbi1Δ mutant, and cbi1ΔC-WT complemented strains were incubated for 24h in YPD+ 10 uM CuSO4 (copper sufficiency) or YPD +250 μM BCS (copper deficiency), harvested, and double stained with CFW and WGA-Alexa 488. Stained cells were analyzed using a FACS Canto A Analyzer, and data were analyzed using Flow Jo. Representative data indicating percentage of total cells staining as “High WGA” (increased exposed chitin/chitosan) after incubation in copper-sufficiency (A) or copper-deficiency (C). Quantification of three independent experiments for copper-sufficiency (B) or copper-deficiency (D) conditions. A 2-way ANOVA was performed from log transformed data using GraphPad Prism. (E)-(F) Cell wall analysis was performed for the WT, cbi1Δ mutant, and cbi1ΔC-WT complemented strains after incubation for 24h in CO2-independent medium at 37°C. Cells were analyzed microscopically using WGA-Alexa 488 staining (E), or by FACS analysis for High WGA cells per above (F). (G) Macrophage activation assay upon infection with copper sufficient or deficient WT, cbi1Δ and cbi1ΔC-WT complemented cells. BMMs were harvested from A/J mice and co-incubated with Cn cells at an MOI of 10:1 (Cn:BMMs), followed by an ELISA-based quantification of TNF-α (pg) in the supernatant. Presented is the mean +/- SEM of 5 independent experiments. A 2-way ANOVA was performed from log transformed data using GraphPad Prism. (I-J) Five-fold serial dilutiuons of indicated strains were incubated on YPD medium supplemented with indicated amounts of BCS (copper starvation) (I) or CuSO4 (toxic copper stress) (J). Plates were incubated at 30°C for 2-6d.
Fig 6.
Cbi1 depletion and copper deficiency impacts cell wall-associated virulence phenotypes.
(A) Minimal inhibitory concentration (MIC) analysis of caspofungin during copper deficiency. WT, cbi1Δ and cbi1ΔC-WT strains were grown in 96-well liquid cultures in SC media supplemented with 100 μM BCS and were serial dilutions of caspofungin. OD600 was measured after 24h of growth at 30°C and the OD600 of the non-treated condition was set to 1. Presented is the mean +/- SEM of 4 independent experiments. A 2-way ANOVA was performed from log transformed data using GraphPad Prism. (B) Melanization of WT, cbi1Δ, ctr1Δ and ctr4Δ cells in the absence and presence of 5 μM CuSO4. Overnight cultures were harvested, washed 1x with PBS, diluted to OD600 2.5 and spotted on to L-DOPA plates to induce melanin production. Shown are representative images from 3 independent experiments. (C) Analysis of capsule formation using India ink counter-staining of WT, cbi1Δ, and cbi1ΔC-WT complemented cells. Indicated strains were grown for 3d in capsule inducing conditions in presence and absence of 250 μM BCS. Cell were harvest, resuspended in PBS, stained with India ink (1,1) and analyzed using DIC optics. Shown are representative images from 3 independent experiments. Scale bar 20 μm. (D) Quantification of capsule size from India ink staining. Images taken were analyzed with imageJ/Fiji. A minimum of 170 cells of each strain were analyzed. Data are presented as box and whiskers diagram with indicated median and min/max capsule size measured for the indicated strains. A mixed effect analysis was performed using the log transformed data. (E) Scanning electron microscopy (SEM) analysis of WT and cbi1Δ cells after incubation in CO2-independent medium for 3 days in absence and presence of 250 μM BCS.