The transcription factor Cuf1 regulates cell wall integrity in response to cellular copper levels through the Cbi1-Ctr1 copper uptake complex

A recent study identified the C. neoformans Cuf1-dependent copper regulon in response to both copper deficiency and copper excess [4]. During copper deficiency, the most upregulated Cuf1-dependent transcripts represent genes involved in copper uptake, including those encoding for the high-affinity copper transporters Ctr1 and Ctr4 and the newly identified Cbi1 protein [4]. Previously referred to as Cn Bim1, this protein has been renamed Cbi1 (Cu-binding and release protein 1) to reflect its known biochemical activities [6,7], and to recognize the previously described Cn Bim1 microtubule-binding protein involved in filamentous growth [16]. Besides genes involved in classical copper homeostasis pathways, this study identified many genes within the Cn Cuf1-copper regulon with functions associated with maintenance of the fungal cell surface and cell wall [4,5] (S1 Table). Among these copper regulated cell wall synthesis genes were two of the eight known chitin synthase genes (CHS5, CHS7), the chitin deacetylase 2 gene (CDA2), and three β-1,3-glucan modulating genes (CNAG_05458, CNAG_05138, CNAG_06636), directly connecting Cuf1-driven transcriptional regulation of copper homeostasis to cell wall remodelling and synthesis. Therefore, to test the effects of Cn Cuf1 function on the fungal cell wall, we cultured the WT strain and the cuf1Δ mutant in presence of high Cu or low Cu stress (induced by the addition of the Cu⁺-chelator bathocuproinedisulfonic acid (BCS)). We then analyzed the strains microscopically after staining with calcofluor white (CFW), a dye binding to the inner cell wall layer formed by chitin and chitosan [17] (Fig 1A and 1B). In both copper stress conditions we observed a decrease in CFW staining intensity in cuf1Δ mutant cells, suggesting lower chitin cell wall content.

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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.

https://doi.org/10.1371/journal.ppat.1010195.g001

The fungal cell wall is a dynamic structure required for viability, stress resistance, morphogenesis and virulence. Its composition is actively remodeled in response to various stress signals, and this process is controlled by conserved signaling cascades, including the cell wall integrity (CWI) pathway [18]. The cell wall stress experienced by the copper homeostasis mutants, specifically during copper deprivation, is reflected in transcriptional alterations in the CWI pathway. The ROM2 gene encodes a guanine nucleotide exchange factor required for CWI pathway activation under conditions of cell stress [19]. Transcriptional induction of ROM2 is therefore typically observed during conditions of cell wall stress. Comparative transcriptional analysis of the WT and cuf1Δ mutant revealed similar ROM2 transcript levels between these two strains during copper sufficiency (Fig 2A). In contrast, ROM2 transcript levels were 3-fold higher in the cuf1Δ strain compared to wildtype during copper starvation. A similar trend was observed for other components of the cell wall stress pathway such as CRZ1 and ROM20 (S1A and S1B Fig). Complementation of the cuf1Δ mutant with the CUF1-FLAG gene (cuf1Δ^C strain) restored the transcript levels to wildtype levels (Figs 2A and S1A and S1B). Taken together depletion of Cn Cuf1 directly effects the fungal cell wall in modulating the inner chitin layer (quantified through CFW staining) and causes increased cell wall stress specifically during copper deficiency.

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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 cuf1Δ^C 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 (cbi1Δ^C-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.

https://doi.org/10.1371/journal.ppat.1010195.g002

To further analyze the impact of CUF1 and other Cuf1-regulated copper homeostasis genes for maintaining cell wall integrity during copper deficiency, we assessed the sensitivity of the cuf1Δ, cbi1Δ, ctr1Δ and ctr4Δ strains to cell wall stressors in copper-replete and copper-deficient conditions. These stresses included calcofluor white (CFW, blocks chitin assembly), Congo red (impairs assembly of cell wall polymers, mainly chitin), caffeine (impacts PKA-mediated signal transduction), SDS (cell surface/ membrane stressor) and NaCl (osmotic stressor) [20–22]. We did not observe any growth phenotypes for the mutant strains in the presence of these cell wall stressors under copper-replete conditions (S1C Fig). In contrast, during copper starvation induced by the extracellular copper (I) chelator bathocuproinedisulfonic acid (BCS) (100μM), the cuf1Δ and cbi1Δ strains exhibited strong growth inhibition compared to WT on NaCl, SDS, Congo red and CFW-containing media (Fig 2B). Even at more modest levels of copper chelation (10 μM BCS), the cuf1Δ and cbi1Δ strains exhibited growth inhibition on Congo red-containing media (Fig 2C). Additionally, the Ctr1 copper transporter was similarly required for survival during copper deprivation in the presence of NaCl and CFW, but not in the presence of SDS. No cell wall stress-associated growth defect was noted for the ctr4Δ copper transporter mutant strain (Fig 2B), supporting prior observations that the Ctr1 and Ctr4 high affinity copper transporters serve overlapping but non-redundant functions in Cn copper homeostasis [23]. Growth inhibition of the cuf1Δ and cbi1Δ strains was complemented by expressing epitope-tagged versions of these proteins in the respective mutant strains [Cuf1-Flag in cuf1Δ (cuf1Δ^C) or Cbi1-HA in cbi1Δ (cbi1Δ^C-WT-HA)]. These results suggest an interaction between copper abundance/homeostasis and Cn cell wall stress resistance.

To further explore the relationships among the Cn copper homeostasis proteins in cell wall integrity, especially the less well-characterized Cbi1 protein, we conditionally overexpressed the CBI1 gene in a series of individual and double mutant strains (Fig 2C). Galactose-mediated overexpression of the pGAL7-CBI1 allele fully complemented the copper-dependent cell wall growth defect of the cbi1Δ mutant, and partially that of the cuf1Δ strain. In contrast, overexpression of CBI1 was unable to restore cell wall integrity to strains with a ctr1Δ mutation. These results are consistent with prior studies suggesting that Cbi1 and Ctr1 are components of a common copper transporter complex [5]. Additionally, these findings indicate that Cuf1 controls many Cu-regulated cell wall processes, but that defective Cbi1 function is likely responsible for much of the loss of cell wall integrity in the cuf1Δ mutant.

We also assessed Cn copper-dependent cell wall integrity phenotypes using an alternative method of copper limitation to extracellular copper chelation by BCS. We incubated the Cn strains in media containing ethanol and glycerol as sole carbon sources. Growth on these non-fermentable carbohydrates is only supported by cellular respiration, effectively shunting intracellular copper into the mitochondria to the copper-dependent enzymes required for oxidative phosphorylation. Incubation of the WT and cbi1Δ^C-WT strains in YPEG + 0.01% SDS caused a ~40% growth reduction, and an even more severe reduction of growth (~90%) in the cbi1Δ strain (Fig 2D). This growth impairment was complemented in all strains by supplementation with CuSO₄, suggesting that shuttling of copper from other pathways towards respiration influences the ability of Cn to withstand cell surface stress. Depletion of Cbi1 further decreased cell fitness under these conditions.

We also tested the cell wall integrity of the Cn ccc2Δ mutant, a strain defective in copper transport withinin the secretory pathway and subsequent altered metalation of secreted proteins [2,24]. A modest growth defect on Congo red was observed for the ccc2Δ strain, and the defect developed independent of copper availability (Fig 2E). These results suggest that defective copper loading of enzymes in the secretory compartment is likely not the cause of the most prominent Cn cell wall phenotypes observed during copper deficiency.

The Cfo1 ferroxidase is a copper-dependent enzyme involved in high-affinity iron acquisition [25]. Therefore, loss-of-function mutations in Cuf1 or other components of the Cn copper uptake machinery would be predicted to affect intracellular iron concentrations as well as copper levels. Additionally, previous studies demonstrated that iron homeostasis is important for proper fungal cell wall and membrane architecture [26,27]. We therefore analyzed the effects of exogenous copper or iron on the BCS-induced cell wall phenotypes of the cbi1Δ and cuf1Δ mutants. Individual supplementation of the growth medium with copper, but not iron, restored growth to the cbi1Δ and cuf1Δ strains in the presence of cell wall stress and copper depletion (Fig 2F). This finding suggest that the observed cell wall stress phenotype is not solely the effect of iron starvation caused by a decrease in copper availability but directly linked to copper limitation.

Changes in Cn cell wall composition in response to defective copper homeostasis

To further characterize the specific role of Cbi1 in cell wall homeostasis during copper stress, we used transmission electron microscopy (TEM) to characterize the cell wall architecture of the wildtype (WT) and cbi1Δ strains incubated in both copper-sufficient and copper-deficient growth conditions (Fig 3A–3C). In copper-sufficient conditions the Cn WT cell wall consists of two layers characterized by differing electron density [28–30]. Extracellular copper sequestration by the highly copper-specific chelator BCS resulted in decreased electron density in the innermost cell wall layer composed primarily of chitin and chitosan (Fig 3A and 3C). These chito-oligomers efficiently bind divalent metals (including copper ions), consistent with the higher electron density of this cell wall layer during copper sufficiency [14,15]. The cell wall of the cbi1Δ mutant strain was similar to WT during copper sufficiency, displaying distinct layers based on electron density. However, the copper-starved cbi1Δ strain demonstrated a reduction in total cell wall thickness compared to both the WT strain and the copper-sufficient cbi1Δ cells (Fig 3B). Also in contrast to WT, there was no reduction in the inner cell wall electron density in the cbi1Δ mutant strains during copper deficiency (Fig 3A and 3C).

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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.

https://doi.org/10.1371/journal.ppat.1010195.g003

The ultrastructural changes observed in the cbi1Δ strain cell wall during copper starvation were not due to altered cell viability. Although the cbi1Δ strain demonstrated a defect in proliferation during copper deficiency, we observed no decrease in cbi1Δ cell viability during the first 24 hours, as assessed by quantitative culture (S1D and S1E Fig). We also assessed the effective “copper state” of the WT and cbi1Δ strains in the conditions chosen for copper sufficiency (10 μM CuSO₄) and deficiency (250 μM BCS) by quantifying transcript levels of the CMT1 metallothionein gene (induced during high copper states) and the CTR4 copper transporter gene (induced during copper starvation) (S1F Fig). Both the WT and cbi1Δ strains demonstrated a strong induction of CTR4 expression, but not of the CMT1 transcript, indicating effective induction of copper deficiency by BCS treatment compared to the copper sufficient growth conditions.

We performed ICP-MS-based metal quantification of cell-associated copper and iron both WT and cbi1Δ strains in copper sufficient and deficient conditions (Fig 3D and 3E). As predicted by the role of Cu-depending proteins in cellular iron import, both WT and cbi1Δ cells show a similar pattern of reduced cell-associated copper and iron after BCS incubation. Therefore, the different pattern in electron density observed in copper starved WT and cbi1Δ cells cannot simply be explained by changes in total cell copper or iron levels. However, this type of ICP-MS analysis of total cellular metals is not sufficiently sensitive to discriminate between cell surface associated and intracellular metal accumulation in these small fungal cells.

To better investigate surface adsorption of copper by different Cn strains, we used anodic stripping voltammetry (ASV) with a Hanging Mercury Drop Electrode. ASV measures copper speciation based on reducibility at an electrode surface [31]. We expected Cu²⁺ adsorbed to the cell surface to be electrochemically inert due to Cu²⁺ chelation by cell wall polysaccharides which makes it kinetically inert for reduction and slow to diffuse to the electrode surface. Using two concentrations (10⁵ or 10⁷ cells/mL) of fungal cells in suspension in PBS, we observed a measurable adsorption of Cu²⁺ onto the Cn cell surface at the higher cell concentration compared to cell-free PBS. These finding shows that Cn cells readily adsorb Cu²⁺ ions on there cell surface with no major differences in adsorption capacity between the WT and cbi1Δ cell surface.

Transcriptional changes in cell wall genes in response to copper status

Prior investigations have established a set of C. neoformans genes encoding enzymes involved in the synthesis and chemical modification of the major cell wall structural carbohydrates. These include 8 chitin synthetase genes (CHS1-8), 4 chitin deacetylase genes (chitin-to-chitosan conversion) (CDA1-3, FPD1), and the chitin synthase regulator-2 (CSR2). Genes involved in glucan synthesis include KRE6 and SKN1 (β-1,6-glucan), FKS1 (β-1,3-glucan) and AGS1 (α-1,3-glucan) [8]. To investigate how copper availability and Cbi1 might affect cell wall carbohydrate homeostasis, we measured transcript levels of these major cell wall synthesis genes in copper deficient wildtype (WT), cbi1Δ mutant, and cbi1Δ^C-WT complemented strain (Fig 4A and 4B). Among the genes involved in chitin and chitosan synthesis, we observed significant increases in transcript abundance in the cbi1Δ mutant for CHS6 (~7 fold), CHS3 (~4 fold) and CSR2 (~3 fold), while the chitin/chitosan deacetylase genes CDA2 and FPD1 were downregulated (Fig 2D). In addition to genes involved in chitin/chitosan biosynthesis, the transcript level of the β-1,3-glucan synthase FKS1 gene was induced 8-fold. Changes in the expression of KRE6 (β-1,6-glucan synthesis) and AGS1 (α-1,3-glucan synthesis) were not statistically significantly altered (Fig 4B). These findings indicate that the copper-deficient cbi1Δ strain displays transcriptional changes in several cell wall polysaccharide synthesis genes, especially those associated with chitosan and β-1,3-glucan synthesis.

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Fig 4. Cbi1-dependent cell wall gene transcript abundance and cell wall chitosan levels.

(A)-(B) The WT, cbi1Δ mutant, and cbi1Δ^C-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 cbi1Δ^C-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.

https://doi.org/10.1371/journal.ppat.1010195.g004

To explore the functional relevance of changes in Cn cell wall gene transcript abundance as a function of copper availability, we quantified β-glucan and chitin/chitosan levels in the WT, cbi1Δ mutant, and cbi1Δ^C-WT complemented strains after incubation for 24h in copper-sufficient (YPD + 10 μM CuSO₄) and copper-deficient (YPD + 250 μM BCS) conditions. No significant changes were detected in total cell wall β-glucan between the WT and cbi1Δ cells in either growth condition (S2A Fig). However, the cbi1Δ mutant exposed to copper-deficiency displayed a greater than 50% reduction in total cell wall chito-oligomers due to a drastic reduction of chitosan compared to WT and complemented strains (Fig 4C and 4D). Therefore, the reduction of cell wall thickness and altered cell wall integrity in the cbi1Δ strain during copper starvation is, in part, likely due to a reduction in the inner cell wall chito-oligomer layer, especially chitosan.

In addition to measuring alteration in cell wall polysaccharide content, we also examined changes in the patterns of chitin and chitosan deposition using chitin- and chitosan-specific fluorescent stains. We double-stained Cu-sufficient and Cu-deficient Cn cells with Calcofluor white (CFW), a small molecule staining 1,3-β-and 1,4-β-polysaccharides of chitin/chitosan [17], and AlexaFluor488-conjugated wheat germ agglutin (WGA-Alexa 488), a lectin that binds exposed chito-oligomers [32,33]. Consistent with the reduction in total cell chitin/chitosan measured in the biochemical assays, we observed a similar reduction in CFW staining intensity of Cu-deficient cbi1Δ cells (Figs 4E and S2B). WGA staining of WT and cbi1Δ^C-WT complemented cells revealed exposed chito-oligomers primarily at regions of cell separation, budding sites, and bud scars. In contrast, the copper-deficient cbi1Δ cells demonstrated an enrichment of WGA-Alexa 488 staining globally around the cell surface. Our biochemical analysis of the cbi1Δ cell wall composition showed that the reduction in total chitin was primarily due to significant loss of chitosan. Therefore, to specifically stain for chitosan we used EosinY, a small fluorescent molecule that binds to chitosan polymers, for further microscopic analysis of Cu-sufficient and Cu-deficient Cn cells (Figs 4F and S2C and S2D). Consistent with the biochemical data, we observed decreased EosinY staining intensity in copper-starved cbi1Δ cells.

We also performed flow cytometry on CFW- and WGA-Alexa 488-stained cells to more precisely quantify the altered chitin/chitosan staining pattern in these strains (Figs 5A–5D and S2E and S2F). We directly compared WGA-Alexa 488 staining intensity to CFW staining intensity to assess chito-oligomer exposure relative to total cell wall chito-oligomer content. In copper-sufficient growth conditions, there was a small but statistically significant decrease in WGA-Alexa 488 staining intensity of the cbi1Δ cells compared to WT and complemented strains. However, we observed a notable increase in relative WGA staining in a sizable subpopulation of cbi1Δ cells (“High WGA”, Figs 5C and 5D and S2E) during copper-starvation, consistent with the fluorescent microscopy results.

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Fig 5. Cbi1 depletion effects chitin architecture during copper deficiency and host stress.

(A)-(D) The WT, cbi1Δ mutant, and cbi1Δ^C-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 cbi1Δ^C-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 cbi1Δ^C-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.

https://doi.org/10.1371/journal.ppat.1010195.g005

Upon infection, Cn has to react to and withstand a combination of abiotic and biotic stresses, including metal stress. To investigate whether the observed changes of the cell wall architecture in the cbi1Δ mutant are also induced under host-like stress conditions, we cultured WT, cbi1Δ, and cbi1Δ^C-WT cells for 24h in CO₂-indepedent medium (CIM) at 37°C and performed microscopy and FACS analysis of CFW- and WGA-Alexa 488-stained cells (Fig 5E and 5F). Similar to cbi1Δ cells grown in copper-deficient YPD medium, we observed an increase in WGA-Alexa 488 staining signal in a significant subpopulation of cbi1Δ mutant cells after incubation in CIM.

One potential physiological consequence of aberrant cbi1Δ cell wall architecture is increased activation of host innate immune cells, as previously reported with other Cn strains with increased chitin and chitosan exposure [34,35]. We co-incubated the fungal strains with murine bone marrow-derived macrophages (BMM), assessing TNF-α production as a marker of host immune cell activation. Macrophages exposed to copper-deficient cbi1Δ cells showed a statistically significant increase in TNF-α secretion compared to macrophages co-incubated with similarly treated WT or complemented strains (Fig 5G). No differences in TNF-α production were noted for macrophages incubated with any of these strains grown in the presence of copper. Together, these findings suggest that copper-deficient cbi1Δ cells not only have a decreased total cell wall chitin and chitosan content, but also that these polysaccharides are deposited in an aberrant manner within the cell wall leading to a higher degree of exposure in a manner that is detectable by host immune cells.

To explore the role of chitosan for modulating the resistance to copper stress, we assessed the growth effects during low and high copper stress for strains with mutations in either copper homeostasis or chitosan synthesis (Fig 5H and 5I). As previously described, we observed poor growth of the cbi1Δ and ctr1Δ strains during copper deficiency (Fig 5H). In line with previous findings, no sensitivity to copper starvation was observed for the ctr4Δ copper transporter mutant strain [36]. These results indicate that the Cbi1/Ctr1 complex can likely compensate for the loss of Ctr4, but that the Ctr4 transporter is not sufficient to maintain Cn growth during copper starvation in the absence of Ctr1. A slight reduction in growth was observed for the chitosan-deficient chs3Δ strain during copper starvation. Chs3 is responsible for the synthesis of most of the chitin destined to be converted to chitosan, suggesting that reductions in chitosan by varied mechanisms may affect the ability of Cn to withstand low copper stress. No growth phenotype was observed for the cda2Δ chitin deacetylase gene, indicating that the observed dysregulation of CDA2 in the cbi1Δ background does not fully explain the cbi1Δ growth defect during copper deficiency.

To assess the role of cell wall chitin and chitosan in resistance to copper toxicity, we tested the chs3Δ and cda2Δ mutant strains for growth in the presence of increasing copper concentations (Fig 5I). The chitosan-deficient chs3Δ strain was more sensitive to high copper stress than the wild-type, and similar in its copper sensivity to strains with mutations in the Cn metallothionein genes CMT1 and CMT2 that mediate scavenging of excess copper. In contrast to chs3Δ, the cda2Δ strain, with a mutation in a single chitin deacetylase gene but relatively preserved cell wall chitosan levels, demonstrated increased resistance to toxic copper levels compared to WT. The cbi1Δ mutant displayed a similar copper resistance profile as the cda2Δ strain. This increased copper resistance was not shared with the ctr1Δ copper transporter mutant and and less apparent in the ctr4Δ mutant strain, suggesting that altered copper transport alone was not responsible for this phenotype. These findings suggest an unexpected new role for Cbi1 in modulating growth during high copper stress.

Cell wall changes in response to low copper stress lead altered caspofungin tolerance, melanin deposition and capsule architecture

Compared to many fungal pathogens, C. neoformans is relatively tolerant to caspofungin, an antifungal drug that inhibits β-glucan synthesis. However, C. neoformans strains with defects in chitosan production are more susceptible to this drug [37]. This observation is consistent with a conserved compensatory increase in cell wall chitin and chitosan among diverse fungi upon treatment with caspofungin [38,39]. Given the reduced cell wall chito-oligomer content in the copper-starved cbi1Δ mutant, we hypothesized that this strain would be more susceptible to caspofungin. Consistent with this hypothesis, we observed an 8-fold decrease in the effective inhibitory dose for 50% growth inhibition (ED₅₀) for caspofungin for the cbi1Δ mutant strain compared to the WT and reconstituted strains when incubated in copper-deficient conditions (MIC₅₀ cbi1Δ 3.1 μM) (Figs 6A and S2G). This suggests that the fungal cell wall mediates durable and adaptive cellular responses to copper availability that affect antifungal drug activity.

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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 cbi1Δ^C-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 cbi1Δ^C-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.

https://doi.org/10.1371/journal.ppat.1010195.g006

The cryptococcal capsule is composed of highly branching polysaccharides that are covalently attached to components of the outer surface of the cell wall, especially α-1,3 glucans [40]. The thickness of the capsule is induced during incubation conditions that mimic the host environment, including slightly alkaline pH and micronutrient limitation [11]. Strains with mutations in several chitin deacetylase genes, in particular with a cda2 deletion, typically display enlarged capsules [22]. Similarly, the cbi1Δ mutant, in which CDA2 levels are reduced, displayed more surface capsule, especially in copper limiting conditions (Fig 6C and 6D).

We performed scanning electron microscopic (SEM) analysis of WT and cbi1Δ cells after 3d of capsule induction in the absence and presence of 250 μM BCS (Fig 6E and 6F) to further analyze the role of Cbi1 in capsule architecture with a higher topographical resolution. Similar to capsule assessment by India ink counterstaining, we observed the most notable alterations in the capsular structure of the cbi1Δ strain during copper limitation. Under normal capsule-inducing conditions, both strains showed typical capsule architecture with a dense inner zone and a less dense outer layer consisting of extended capsule fibers [40]. However, when copper deficiency was induced by BCS supplementation, the cbi1Δ capsule displayed denser and more interconnected polysaccharide fibers compared to WT, with fewer freely extending individual capsule fibers.

Both copper limitation and cell wall alterations are important for the regulation of melanin, a cell wall-associated antioxidant pigment [24,30]. Melanin production itself is tightly linked to cellular copper levels since the rate-limiting phenoloxidase enzyme involved in melanin synthesis, laccase-1 (Cn Lac1), is functionally dependent on copper [41]. The cbi1Δ strain displayed defective melanin production on L-DOPA containing media (Fig 6B). The restoration of normal melanin production by the addition of 5 μM CuSO₄ is consistent with the known defects in copper acquisition in this strain [5]. We did not observe altered melanin production for the ctr1Δ or ctr4Δ strains, suggesting that the degree of intracellular copper limitation is greater in the absence of of the Cbi1 protein than in either of the single copper transporter mutants. Also, chitosan is required for the incoporation of melanin within the cell wall, and strains completely lacking chitosan display a “leaky melanin phenotype” in which melanin diffuses from the cell into the growth medium [22]. Even though depletion of Cbi1 affects cell wall chitosan levels during copper deficiency, we did not observe a leaky melanin phenotype. This is consistent with a reduction but not the complete absence of chitosan in the cbi1Δ strain. Together these results suggest that Cbi1-mediated cell wall remodeling and copper homeostasis affect the establishment of several cell wall-associated virulence factors.

Ethics statement

All animal experiments in this manuscript were approved by the Duke University Institutional Animal Care and Use Committee (IACUC) (protocol #A102-20-05). Mice were handled according to IACUC guidelines.

Strains, media and growth conditions

Cryptococcus neoformans strains used in this study are shown in S2 Table. All strains were generated in the C. neoformans var. grubii H99 background. For strain creation, DNA was introduced into C. neoformans by biolistic transformation [58]. Yeast extract (2%)-peptone (1%)-dextrose (2%) (YPD) medium supplemented with 2% agar and 100 μg ml-1 of nourseothricin (NAT), 200 μg ml-1 of neomycin (G418) or 200 μg ml-1 of hygromycin B (HYG) was used for colony selection after biolistic transformation. Cloning strategies as well as plasmids and oligos used for creation of Cryptococcus transformation constructs are described in S3 and S4 Tables Transformants were screened by PCR and Southern blot for intended mutations. Cbi1-HA expression among relevant transformants was confirmed by western blot.

Strains were cultivated in either synthetic complete (SC) medium (MP Biomedicals) or YPD at 30°C. To induce Cu sufficiency or deficiency, media was supplemented with indicated concentrations of CuSO₄ or the Cu⁺ chelator bathocuproine disulfonate (BCS), respectively. Alternatively, to BCS supplementation, strains were cultivated in Yeast extract-peptone medium supplemented with 3% Glycerol and 2% Ethanol (YPEG). For galactose-regulated expression induction, SC+2% Galactose (SC-Gal) or SC+2% Glucose (SC-Glu) was used and supplemented as indicated. To analyze cell wall associated phenotypes, caffeine (0.5 mg/ mL), NaCl (1.5 M), SDS (0.01%), Congo red (0.5%) and Calcofluor White (1.5 mg/mL) were added to SC medium supplemented with CuSO₄ or BCS as indicated. For growth phenotype analysis on solid medium plates, a 6-fold serial dilution, starting at OD₆₀₀ 0.25, of strains was spotted and incubated for indicated time and temperature. For assessment of melanization, overnight cultures in YPD were washed once in PBS and resuspended in PBS to OD₆₀₀ 2.5. Next, 5 to 10 μL of the resuspended culture were spotted onto L-3,4-dihydroxyphenylalanine (L-DOPA) media (7.6 mM L-asparagine monohydrate, 5.6 mM glucose, 22 mM KH2PO4, 1 mM MgSO4.7H2O, 0.5 mM L-DOPA, 0.3 μM thiamine-HCl, 20 nM biotin, pH 5.6). L-DOPA plates were incubated at 30°C for 2 days. To induce capsule, strains were incubated in CO2-independent tissue culture medium supplemented as indicated (TC, Gibco) for 72 hours with shaking at 37°C, followed by staining with India Ink or fixation for scanning electron microscopy (SEM).

RNA isolation and qRT-PCR

For ROM2 transcript analysis C. neoformans overnight cultures grown in synthetic complete (SC) medium (MP Biomedicals) were diluted to OD₆₀₀ 0.3, and cultures were supplemented and cultivated as indicated. For cell wall synthesis genes transcript analysis and copper status analysis, C. neoformans overnight cultures grown in YPD medium were diluted to OD₆₀₀ 0.05, supplemented as indicated and cultivated for 24h at 30°C. For RNA extraction, cells were cultivated as indicated. Cultures were harvested, washed 1x with PBS and flash frozen on dry ice, followed by lyophilization. RNA was extracted using the RNeasy Plant Mini Kit (Qiagen) with optional on-column DNAse digestion. cDNA for real time-PCR (RT-PCR) was prepared using the Iscript cDNA synthesis kit (Biorad). For RT-PCR, cDNA was diluted 1:5 in RNase-free water, added to ITAQ Universal SYBR Green Supermix (Bio-Rad) per protocol instructions and analyzed on a CFX384 C1000 ThermoCycler (BioRad, ROM2 analysis and Cu status analysis). For analysis of cell wall transcripts, the diluted RNA was mixed with the PowerUP SYBR Green Master mix (applied biosystems) per protocol instruction and analyzed on a QuantStudio 6 Flex (applied biosystems). Oligos used for qRT-PCR analysis are shown in S4 Table. C_T values were determined using the included CFX Maestro software (BioRad) or the QuantStudio 6 Flex, respectively. Gene expression values were normalized to the housekeeping gene GAPDH and expression fold changes determined by the ΔΔC_T method. For all qRT-PCR studies, a minimum of 3 independent biological replicates were used for the analysis of mRNA expression changes.

Inductively coupled plasma mass spectrometry (ICP-MS)

Cell associated metals (Fe and Cu) were quantified from lyophilized Cn cells. In short, yeast cells were treated as indicated, spun down and washed 2x with ICP-MS grade water. In the last wash step, cell were counted, spun down and lyophilized. Samples were digested in 300 uL 50% ICP-MS grade Nitric Acid for 1h, at 90°C and cooled down overnight at RT. Metal content was analyzed by ICP-MS at the Oregon Health Sciences University Elemental Analysis Shared Resource using an Agilent 7700X ICP-MS as published previously [49].

Anodic stripping voltammetry (ASV)

The ASV experiment was performed with live cells. Cells were cultivated in copper deficient conditions (YPD + 250 μM BCS) for 24h at 30°C to remove loosely bound copper on the surface of the Cn cells. Prior to analysis, cells were harvested and washed 3x with 25 mL PBS and resuspended in PBS. Cells were counted twice and normalized to either 10⁵ cells/ mL or 10⁷ cells/mL (in PBS) for analysis. Copper concentration between 250 nM to 1000nM were titrated stepwise into 20 mL cell suspensions and copper adsorption after each titration step was measured using a static mercury drop electrode (663VA Stand and PGSTAT 128N potentiostat, Metrohm-Autolab, drop size 2), an Ag/AgCl, KCl (3 M) reference electrode with a 3M KCl salt bridge, and a glassy carbon counter electrode. Samples were initially purged with highpurity N2 (5.0, Airgas) for 180 s. ASV stripping peak currents were measured after a deposition time of 90s at -1.2 V. After an equilibration time of 5 s, the potential was ramped from −1.2V to 0V using a square wave voltammetry (scan rate 0.12Vs−1; amplitude 0.02V; frequency 60Hz). For analysis the measured curve was smoothed and the peak height at E = -0.12V was determined. For each titration experiment, the peak height was plotted against titrated Cu concentration and a linear regression was performed to calculate the slope (measured current/ nM Cu).

Liquid growth assays

All liquid growth analysis were performed in 96 well plates. For the growth analysis in YPEG in the presence of the surface stressor SDS, overnight cultures (YPD, 30°C), were harvested and washed 1x with YPEG and then normalized to OD₆₀₀ 2.0 (in YPEG). Growth media was supplemented as indicated and filled into 96 well plate (195 μL each well). Wells were inoculated with 5 μL of indicated strain (final OD₆₀₀ = 0.05). Plates were covered with a semipermeable membrane (Breathe-Easy, Diversified Biotech) and incubated at 30°C with shaking at 1150 rpm in a Finstruments shaker instrument. Growth graphs of the indicated strains at the conditions analyzed were generated by plotting the OD₆₀₀ readings normalized to WT-YPEG growth at the 24 h time point. Three biological replicates were performed.

For minimal inhibitory concentration analysis (MIC), overnight cultures (SC, 30°C), were harvested and washed 1x with PBS and then set to OD₆₀₀ 0.25 (in PBS) and stored on ice until further usage. 2x concentrated working stocks of caspofungin were prepared in SC medium (final concentration in assay ranged 100 to 0.78 μg/mL). Cells were diluted 1:100 in either SC or SC+200 μM BCS (final concentration in assay 100 μM BCS). In 96 well plate, 100 μL of diluted cells were mixed with 100 μL of 2x concentrated caspofungin stocks. The plate was covered with a semipermeable membrane (Breathe-Easy, Diversified Biotech) and incubated at 30°C with shaking at 1150 rpm in a Finstruments shaker instrument for 24h. Growth graphs of the indicated strains at the conditions analyzed were generated by calculating the relative growth of the drug-treated condition in relation to the untreated condition (drug-treated OD600/untreated OD600). Four biological replicates were performed.

Transmission electron microscopy (TEM)

Overnight cultures (YPD, 30°C) were harvested and washed 1x with YPD. Indicated strains were inoculated to an OD₆₀₀ of 0.05 in 50 mL YPD + 10 μM CuSO₄ (= Cu sufficiency) or 250 μM BCS (= Cu deficiency) and cultivated for 24h at 30°C. In the following day, 50 to 100 μL of the culture were harvested and washed 1x with PBS. Next, cells were pelleted and overlayed with fixative (4% formaldehyde, 2% Glutaraldehyde in PBS) and incubated for 4h at RT. Then, fixative was removed, and the sample was washed twice with 1x PBS, with a 10 minute incubation time at RT in between wash steps. After last washing step, the PBS was removed and 1% OsO₄ was added to the sample to complete cover. The tube was sealed and incubated for 1h at RT in the dark. Then the OsO₄ was removed, and the sample rinsed with 1x PBS, 2 times for at least 10 minutes each time (RT). After last PBS rinse, residual PBS was removed and the sample was rinsed with 0.1N acetate buffer, 1 time at least 10 minutes each time (RT). The acetate buffer was removed, and the sample was stained with 0.5% uranyl acetate (UA) for one hour, RT. Once staining was complete, the uranyl acetate was removed and the sample rinsed with 0.1N acetate buffer, 2 times at least 10 minutes each time. In the next steps the samples were dehydrated in several ethanol incubation steps by rinsing twice, at least 10 minutes each time, with 30% Ethanol, 50% Ethanol, 70% Ethanol and 90% Ethanol. Finally, the sample were rinsed 3 times, at least 10 minutes each time, with 100% Ethanol.

Once the dehydration was complete, ethanol was removed and the dehydrated sample was embedded into resin (53,5% (w/v) resin, 20.5% (w/v) DDSA, 26% (w/v) NMA, 1.4% (v/v) DMP-30). The sample were incubated in resin mix at RT overnight. The following day, samples were incubated at 50–60°C for 10 minutes, the old resin mix was replaced by freshly made resin mix and incubated for 10 mins at RT, followed by 10 minutes at at 50–60°C. This resin wash step was repeated one more time, followed by a 48h incubation at 50–60°C. The embedded samples were cut into 70nm thick sections on an Ultracut microtome and placed on TEM grids. The sections were counterstained with uranyl acetate and lead citrate and then imaged on an FEI Technai G2 Twin transmission electron microscope. Cell wall thickness was measured using ImageJ (Cu sufficient WT: 8 cells, Cu deficient WT: 7 cells, Cu sufficient cbi1Δ: 14 cells and Cu deficient cbi1Δ: 10 cells). The staining contrast in the cell wall was measured using Image J gray scale measurement tool.

Scanning electron microscopy (SEM)

Overnight cultures (YPD, 30°C) were harvested and washed 1x with PBS. Indicated strains were inoculated to an OD₆₀₀ of 0.1 in 25 mL CO₂-independent medium (Gibco) or 25 mL CO₂-independent medium supplemented with 250 μM BCS and cultivated for 3d at 37°C. 5 mL of each culture were harvested, checked for capsule formation by India ink stain and washed 3x with PBS (without calcium and magnesium). Cells were fixed for 1h at RT with 2.5% glutaraldehyde in PBS, washed 3 times with PBS and checked for intact capsule by india ink staining. Then, cells were mounted onto poly-L-lysine-coated coverslips (Neuvitro, 12mm, #1 thickness coverlsips) and incubated for 20 min at RT. After mounting, cells were sequentially dehydrated in several ethanol washes (1x 30%, 1X 50%, 1X 70%, each 5 min RT, followed by 1x 95% and 2x 100%, 10 min RT). After dehydration mounted cells were stored in 100% ethanol until the critical point drying. Cell samples were critical point dried with a Tousimis 931 critical point dryer (Rockville, Maryland) and coated with gold-palladium using a Cressington 108 sputter-coater (Watford, United Kingdom). Samples were mounted and imaged on a Hitachi S-4700 scanning electron microscope (Tokyo, Japan).

Cell wall isolation and analysis

Overnight cultures (YPD, 30°C) were harvested and washed 1x with YPD. Indicated strains were inoculated to an OD₆₀₀ of 0.05 in 50 mL YPD + 10 μM CuSO₄ (= Cu sufficiency) or 250 μM BCS (= Cu deficiency) and cultivated for 24h at 30°C. The following day, 10 to 25 mL of the cells were harvested and washed twice with dH₂O. In the last wash step, cells were counted, spun down and lyophilized. Chitin and chitosan levels were quantified from lyophilized yeast using a modified MBTH (3-methyl-benzothiazolinone hydrazine hydrochloride) method as previously described [13]. β-glucan was quantified using the megazyme yeast β-glucan kit. In short lyophilized yeast were milled using glass beads, resuspended in 800 μL 2 M KOH and transferred into a new 12 ml reaction tube and stirred for 30 mins in an ice water bath. Then, 3.2 mL of 1.2 M sodium acetate pH 8.3 and 40 μL glucazyme was added and the sample stirred for 2 min. The sample was transferred to a 15 mL screw cap tube and incubated ON at 40°C in a water bath. The next day, 10 mL dH₂O was added to the samples, mixed thoroughly and centrifuged for 10 mins at 3000 rpm. Then, 100 μL of the supernatant was mixed with GOPOD reagent and incubated for 20 mins at 40°C in a water bath. 2 x200 μL of each sample were transferred into a 96 well plate and read (against reagent blank) at 510 nm. A standard curve was prepared using the manufacturer’s supplied D-glucose standard solution and mg glucose was calculated using equation provided by manufacturer. Measured values were normalized by cell count.

Cell wall staining and flow cytometry

Prior to analysis cells were treated as indicated. To visualize chitin, cells were harvested and stained with 100 μg/ml Alexa488-conjugated wheat germ agglutinin (WGA, Molecular Probes) for 35 minutes in the dark, RT, followed by 25 μg/ml calcofluor white (CFW, Fluka Analytical) for 10 minutes, RT. After staining, cells were washed 2x with PBS and were resuspended in 20–50 μL PBS for microscopic analysis. Alexa488-WGA was imaged using a GFP filter and CFW was imaged using a DAPI filter. For flow cytometry fixed or live cells were measured. For Fixation, cells were harvested and washed and then incubated for 5 mins in 3.5% Formaldehyde at RT. Cells were Alexa488-WGA and CFW stained as previously described, washed 2x with PBS and set to 10⁶ cells (in 1 mL PBS). Alexa488-WGA stained cells were analyzed using a 488 nm laser and CFW cells were analyzed using a 405 nm laser. The FACS analysis was performed at the Duke Cancer Institute Flow Cytometry Shared Resource using a BD FACSCanto II flow cytometer. Data was analyzed using FlowJo v10.1 software (FlowJo, LLC). For analysis only single cells were used (gated using the FSC/SSC plot). For chitin exposure analysis cells were gated in the CFW intensity/ Alexa488-WGA intensity scatter plot. Additional histograms with mean fluorescence intensity (MFI) on the x-axis and cell counts on the y-axis were created. Unstained cells were used as negative controls.

To visualize chitosan, cells were treated as indicated, harvested and washed 2x with McIlvaine’s buffer (0.2 M Na2HPO4, 0.1 M citric acid, pH 6.0). Then, cells were stained using 500 μL of 300 μg/ml Eosin Y in McIlvaine’s buffer for 10 minutes at room temperature in the dark. Cells were then washed 2x with McIlvaine’s buffer and resuspended in 20–50 μL McIlvaine’s buffer. Cells were visualized using a GFP filter.

Microscopic quantification

Differential interference microscopy (DIC) and fluorescent images were visualized with a Zeiss Axio Imager fluorescence microscope (64X objectives). Images were taken with an AxioCam MRm digital camera with ZEN Pro software (Zeiss). The same exposure time was used to image all strains analyzed. Images were analyzed using ImageJ/Fiji software. Gray scale values were measured and normalized towards cell count. The intensity of the control strain (= Cu sufficient WT) was set to 1. Results are reported as relative fluorescence intensity +/- standard error of the means.

Cells sizes were measured using the ImageJ measurement tool. Capsule thickness was calculated using the equation:

Generation of bone marrow derived macrophages

Murine bone marrow cells were isolated from A/J mice and prepared as previously described [34]. Briefly, femurs and tibias were isolated from mice. Each bone was flushed with 5 to 10 ml cold PBS using a 27½ gauge needle. Red blood cells were lysed in 1x RBC lysis buffer (0.15 M NH₄Cl, 1 mM NaHCO₃, pH 7.4) and cells were resuspended in 1x Dulbecco’s modified Eagle’s medium (DMEM; + 4.5 g/L D-Glucose, + L-Glutamine, +110 mg/L sodium pyruvate) with 1 U/ml pencillin/streptomycin. Bone marrow cells were cryopreserved in 90% FBS/10% endotoxin-free DMSO at a concentration of 1 x 10⁷ cells/ml.

BMMs were differentiated in BMM medium (1x Dulbecco’s modified Eagle’s medium [DMEM; + 4.5 g/L D-Glucose, + L-Glutamine, +110 mg/L sodium pyruvate], 10% fetal bovine serum [FBS; non-heat inactivated], 1 U/ml penicillin/streptomycin) with 3 ng/ml recombinant mouse GM-CSF (rGM-CSF; R&D Systems or BioLegend)) at a concentration of 2.5 x 10⁵ cells/ml in 150 x 15 mm petri plates at 37°C with 5% CO₂. The media was refreshed every 3–4 days and the cells were harvested after ~7d or when confluency was achieved.

Macrophage co-incubation and TNF-α quantification

Prior to co-incubation, overnight cultures of C. neoformans strains (YPD, 30°C) were harvested and washed 1x with YPD. Indicated strains were inoculated to an OD₆₀₀ of 0.05 in 50 mL YPD + 10 μM CuSO₄ (= Cu sufficiency) or 250 μM BCS (= Cu deficiency) and cultivated for 24h at 30°C. To prepare BMMs for the co-incubation assay, BMMs were counted (by hemocytometer, with Trypan blue to discount dead cells), plated in BMM medium in 96-well plates at a concentration of 5 x 10⁴ cells/well and incubated at 37°C with 5% CO₂ overnight. The next day C. neoformans cells were washed 2x with PBS, counted, and added to BMMs containing 96-well plates at a concentration of 5 x 10⁵ fungal cells per well (10:1 C. neoformans cells:BMMs). Co-cultures were incubated for 6h at 37°C with 5% CO₂. Supernatants were collected and stored at -80°C until analysis. Secreted TNF-α was quantified in supernatants by enzyme-linked immunosorbent assay (ELISA; BioLegend).

Statistical analysis

All data error bars represent statistical errors of the means (SEM) of results from a number of biological replicates (N), as indicated in figure legends. Before statistical analysis was conducted, data from all experiments was log transformed for comparison of proportions. Statistical analysis was performed with GraphPad Prism software v9. The statistical tests chosen for each experiment and their results (i.e., p values) are indicated in figure legends. Asterisks in figures correspond to statistical significance as follows: ****, P < 0.0001; ***, P = 0.0001 to P < 0.001; **, P = 0.001 to P < 0.01; *, P = 0.01 to P < 0.05; ns (not significant), P > 0.05.