Fig 1.
CnCel1 shows homology with LPMOs of the AA9 family.
(A) Phylogenetic tree of lytic polysaccharide monooxygenase (LPMO) families. Sequences of characterized proteins only (Uniprot number or Protein Data Bank (PDB) code provided in the figure) were aligned using T-coffee Expresso [89]. Phylogeny was inferred using PhyML software [86] and the Whelan and Goldman (WAG) amino acid substitution model [87]. Branch support was calculated by 100 bootstrap repetitions (value displayed on tree). The tree was visualized with Interactive Tree Of Life (iTOL) software [88]. Substrate specificities of each LPMO family are shown in grey. (B) Model of CnCel1 (magenta) made using alphafold2 and downloaded from Uniprot [91]. The model is aligned to TaAA9A (PDB: 2YET) (green). The modelled surface of CnCel1 is shown in light grey. CnCel1 aromatic residues potentially involved in substrate binding are shown as stick representation (blue). The histidine brace and the apical tyrosine align. The CnCel1 C-terminal region is shown shortened and in red (C) Phyre2 model of CnCel1 (cyan). The CnCel1 surface is shown in gray. The model is aligned to TaAA9A (PDB: 2YET) (green). CnCel1 aromatic residues potentially involved in substrate binding are shown as stick representation (blue). The CnCel1 dCTR region is shown shortened and is highlighted in red. (D) Progress curves for oxidation of reduced fluorescein by Cel1cat. As positive control the oxidation of reduced fluorescein by 1 μM copper loaded-TaAA9A is shown. Activity measured by relative fluorescent units (RFU) at 528 nm for 30 min. Reaction conditions are copper loaded enzyme concentrations as indicated, 75 mM phosphate citrate, pH 7.4, 25°C (black line). 100 μM dehydroascorbic acid (red line), 100 μM H2O2 (blue), or both (green line) were added as co-substrates to investigate Cel1cat redox cycling propensity. Experiments done in triplicates, standard deviations shown, but not visible.
Fig 2.
CEL1 is induced during alkaline pH and high temperature stress in a Rim101-dependent manner and is associated with the Cn cell wall.
(A) Cel1-4xFLAG protein expression assessed by western blot. The strain expressing the Cel1-4xFLAG fusion protein was incubated in SC medium (starting OD600 of 0.4) for 3h at indicated pH and temperature, or in SC medium supplemented with 1mM CuSO4 or 1mM BCS as indicated. Protein extraction was performed by TCA-based protein extraction. For each condition, 20 μL of crude TCA extract was analyzed by Western blot for Cel1-4xFLAG using the αFLAG-HRP conjugate antibody. The αH3 (anti-histone 3) antibody was used as a loading control. (B) Transcript abundance of Cn CEL1, CAP60, and two other predicted Cn LPMOs (CNAG_03405 and CNAG_07314). Previously reported RNASeq data was analyzed to quantify alterations in the expression of these four genes in response to changes in pH, as well as in two mutants impaired for growth at high pH (sre1Δ and rim101Δ mutant strains): Brown et al., 2020 [29] (WT pH8 vs. pH4 and pH8 sre1Δ vs. WT) and Brown et al., 2018 [28] (Tissue culture (TC) rim101Δ vs. WT). Transcript data were plotted with GraphPad Prism and are shown in log2-fold change. (C) Cel1-4xFLAG protein expression analysis by western blot in response to alkaline pH stress in the WT and rim101Δ strain. The two strains were incubated at 30°C in SC medium pH 8.15 (starting OD600 of 0.4) for indicated times, followed by TCA-based protein extraction. For each condition, 25 μL of crude TCA extract was analyzed by western blot for Cel1-4xFLAG expression using the αFLAG-HRP conjugate and αH3 as loading control. (D) Temperature-dependent Cel1-4xFLAG protein expression at alkaline pH. The strain expressing the Cel1-4xFLAG fusion protein was incubated in SC medium pH 8.15 (starting OD600 of 0.4) for 3h at indicated temperatures, followed by TCA-based protein extraction. For each condition, 25 μL of crude TCA extract was analyzed by western blot for Cel1-4xFLAG expression using the αFLAG-HRP conjugate and αH3 as loading control. (E) Cel1-4xFLAG protein expression in tissue culture medium. Two independent strains expressing the Cel1-4xFLAG fusion protein and the WT strain were incubated in either SC medium at 30°C or in CO2-independent tissue culture medium (CIM) at 37°C for 24 h (starting OD600 of 0.1), followed by TCA-based protein extraction. For each condition, 20 μL of crude TCA extract was analyzed by western blot for Cel1-4xFLAG expression using the αFLAG-HRP conjugate and αH3 as loading control. (F) Western blot analysis of Cel1-4xFLAG secretion pattern. Two independent strains expressing the Cel1-4xFLAG protein were incubated in CIM (starting OD600 of 0.1) for 24h. Cell pellets (P) and supernatant (SN) were harvested, and the supernatant was filtered (0.2μ filter) to remove residual cells. Total protein from each sample was extracted using the TCA-based protein extraction method. For each condition, 20 μL of crude TCA extract was analyzed by western blot for Cel1-4xFLAG using the αFLAG-HRP conjugate and αH3 as loading control. (G-H) Western blot analysis of Cel1-4xFLAG (G) and Sec63-GFP (H) protein levels upon cell wall degradation. Cel1-4xFLAG expression was induced as described in (F). Sec63-GFP was grown overnight in YPD at 30°C and then cultivated for 3h in SC+250μM CuSO4 at 30°C. Cell wall degradation was performed using increasing amounts of Zymolyase (10u, 50 u, 100u, 200u). As control for Cel1-4xFLAG and Sec63-GFP protein levels in the intact cell wall, the assay was performed without the addition of Zymolyase (0u sample). Total protein from each sample was extracted using the TCA-based protein extraction method. For each condition, 20 μL of crude TCA extract was analyzed by western blot for Cel1-4xFLAG using the αFLAG-HRP conjugate (G) and for Sec63-GFP (H) using the αGFP antibody. Histone3 levels were determined using the αH3 antibody.
Fig 3.
cel1Δ shows decreased virulence in two different models of cryptococcosis.
(A) Survival in murine inhalation model of cryptococcosis. Female C57BL/6 mice were infected by inhalation with 105 cells of the indicated strains (10 mice per strain). Mice were monitored twice daily and sacrificed when 15% weight loss was achieved or when exhibiting symptoms linked to imminent mortality. Survival data were plotted, and a log-rank test as statistical analysis was performed using GraphPad prism. (B) Lung colonization at early stages of murine infection. Female C57BL/6 mice (5 mice per strain) were infected with indicated strains as described in (A). At timepoints indicated mice were sacrificed, the lungs were harvested, and lung lysates were assessed by quantitative culture, normalized by lung wet weight. Data were plotted and an unpaired t-test was performed using GraphPad Prism. (C) Lungs of female C57BL/6 mice infected with 105 WT or cel1Δ cells sacrificed at 3 d post infection were harvested for histopathological analyses. Hematoxylin and eosin staining was used to visualize microscopic lung pathology. Fungal cells are indicated with yellow arrowheads. The area used for 20x magnification is marked by a yellow box. (D-E) Lethal infection study in larvae of the Greater wax moth G. mellonella. Larvae were infected by injection of 107 cells of indicated strains (30 larvae per strain). Infected larvae were incubated at 30°C (D) or 37°C (E) and monitored daily for viability (movement and melanin production) and pupae formation. Log-rank test was performed using GraphPad prism. Pupae formation events are marked as an “0” within the survival curve.
Fig 4.
CnCel1 affects cell wall integrity under high temperature and alkaline pH stress.
(A-B) Growth analysis in the presence of cell wall/ surface stressors and high temperature (A) or alkaline pH (B) stress. Five-fold serial dilutions of cell suspensions for each strain were incubated on YPD medium (-) or pH-buffered YPD medium at 30°C or 37°C for 4-7d as indicated in the figure. Cell wall stressors were supplemented to the YPD medium as indicated. (C) Microscopic analysis of CFW and WGA-Alexa-488 stained cells. The WT and cel1Δ strains were incubated overnight in YPD medium and resuspended to OD600 0.1 in indicated medium (either YPD or YPD pH 8.15). Cells were cultivated for 24h hours at indicated temperatures and stained with CFW (total cell wall chitin) and WGA-Alexa488 (exposed cell wall chitin). Shown are representative images of this analysis. The CFW signal (blue channel) and WGA-Alexa 488 (green channel) are presented as merged images. Cell wall quantification of (D) total chitin and (E) β-glucan. Indicated strains were incubated for 24h in YPD 30°C, YPD pH 8.15 30°C or YPD 37°C. Cell wall material was purified from lyophilized yeasts, and total chitin was quantified using the MBTH-based chitin/chitosan quantification method. β-glucan was quantified from lyophilized yeast cells using the Megazyme yeast β-glucan kit. Data represent the mean +/- SEM of 4 biological replicates. A 2-way ANOVA was performed from log-transformed data using GraphPad Prism.
Fig 5.
CnCel1 affects melanization and capsule architecture.
(A) Melanization in the absence and presence of exogenous copper. Indicated strains were incubated on L-DOPA plates at 30°C or 37°C, with and without supplementation with exogenous CuSO4. Melanin formation was assessed at 1–2 days as indicated. (B-E) Surface capsule formation and architecture. The WT and cel1Δ mutant were incubated for 3 days in CIM tissue culture medium to induce capsule. Surface capsule characteristics were assessed by (B) India ink counter-staining and (C) SEM. Representative images are shown from 3 independent experiments. Quantification of capsule size (D) and cell body size (E) was performed using ImageJ/Fiji from static images of india ink-stained cells. Mean values from >100 cells per sample (+/- SEM) from 3 biological replicates are shown as bar graph. Data were plotted, and an unpaired t-test was performed using GraphPad Prism. (F) Electromobility of exopolysaccharide. The WT and cel1Δ mutant were incubated in CIM for 3 days. Exopolysaccharide in the culture supernatant was assessed for relative size and gel motility by agarose gel electrophoresis, transferred to a Nylon membrane, and probed with anti-GXM antibody 18B7. Representative image of multiple replicates is shown.
Fig 6.
cel1Δ shows cell cycle defects under host like stress conditions.
(A-D) In vitro titanization. The indicated strains were incubated overnight in YNB medium, diluted to OD600 of 0.001 in titan cell-inducing medium (PBS+10% HI-FBS, 37°C, 5%CO2) [49], and further incubated for 24h, 48h, and 72h. Cells were analyzed by microscopy and cell diameter measured using Image J/Fiji. (A-C) Scatter plots and bar charts of cell size distribution are demonstrated for indicated times. All graphs were generated using GraphPad Prism. (D) Representative DIC images of indicated strains after 72h growth in titan cell-inducing conditions. (E) Budding index (ratio of budded:total cells) of indicated strains after 18h of conditioning in YPD at 30°C or CIM at 37°C. A minimum of 300 cells were assessed per strain and condition. Shown is the mean +/- SEM of 5 biological replicates. Data were plotted using GraphPad Prism. A 2-way ANOVA was performed from log transformed data. (F) Average cell size of indicated strains after 18h of conditioning in YPD at 30°C or CIM at 37°C. A minimum of 150 cells was measured per strain and condition. Shown is the mean +/- SEM of 4 biological replicates. Data were plotted using GraphPad Prism. A 2-way ANOVA was performed from log transformed data. (G) Ploidy analysis in host-mimicking conditions. Indicated strains were inoculated to a starting OD600 of 0.1 and incubated for 24h in YPD at 30°C or CIM at 37°C. Cells were fixed and stained with propidium iodine. Propidium iodine staining was quantified by Flow Cytometry. Data were analyzed using the FlowJo software. A representative histogram is shown from 3 independent ploidy analyses.