Candida albicans exhibits heterogeneous and adaptive cytoprotective responses to antifungal compounds
- Department of Anatomy and Cell Biology, Western University, Canada
- Department of Biology, Concordia University, Canada
- Department of Computer Science and Software Engineering, Concordia University, Canada
- Shmunis School of Biomedical and Cancer Research, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Israel
- Department of Chemical and Systems Biology, Stanford University, United States
- Department of Biochemistry, Western University, Canada
Figures
Figure 1 with 2 supplements
Experimental design and initial single-cell profiling.
(A) The time series experiment begins with three replicates of untreated (UT) cells followed by profiling of rapamycin (RAPA), caspofungin (CSP), and fluconazole (FCZ, 2 replicates at day 2, and 3 …
Figure 1—figure supplement 1
Schema for analysis stragegy and measures of gene experssion.
(A) The bioinformatics pipeline begins with the Read1 (left) and Read2 (right) fastq files (Materials and methods, ‘Quality control, basic processing, and normalization of the sc profiles’). Sc …
Figure 1—figure supplement 2
Comparison of single cell and bulk expression profiles.
(A) The upper left and lower right coordinates show the distribution of gene counts in single-cell profiles of fluconazole (FCZ)-treated cells at day 2 (FCZ_2) and day 3 (FCZ_3) respectively. Counts …
Figure 2 with 1 supplement
Cell-to-cell heterogeneity in untreated (UT) cell populations.
(A, B) Expression levels (VISION scores) of curated signatures for individual UT cells. Cell order is the same in all graphs. (C) Scatterplot of cells (dots) based on expression level after …
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Figure 2—source data 1
Gene signatures related to microbial phenotypic diversity and drug tolerance curated from the literature.
The most variable signatures identified in each analysis are highlighted – dark colors: final selection; light colors: signatures passed the threshold but not selected after filtration (redundancy; non-concordant gene expression; number of most variable genes ≤1).
- https://cdn.elifesciences.org/articles/81406/elife-81406-fig2-data1-v2.xlsx
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Figure 2—source data 2
GFP and RFP oligo primers.
- https://cdn.elifesciences.org/articles/81406/elife-81406-fig2-data2-v2.xlsx
Figure 2—figure supplement 1
Examples of microscopy images of C. albicans cells harboring both a GFP-tagged heat-shock protein 70 (HSP70) and an RFP-tagged TTR1.
Note the mutually exclusive expression: cells usually express either HSP70 or TTR1, but not both.
Figure 3 with 2 supplements
Cell profiles after challenges with different antifungal compounds.
(A) Uniform Manifold Approximation and Projection (UMAP) embedding of all cells including untreated (UT) cells and cells treated with fluconazole (FCZ) (days 2 and 3), rapamycin (RAPA) (day 2), and …
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Figure 3—source data 1
Differentially expressed genes in fluconazole (FCZ)-treated cells classified in the Ribo-dominant (Rd) cluster 1 (pseudo-bulk samples) compared to FCZ-treated cells in the Stress-dominant (Sd) cluster 4 (pseudo-bulk samples).
baseMean = the average of the normalized counts taken over all pseudo-bulk samples; log2FoldChange = log2 fold change between the groups; lfcSE = standard error of the log2FoldChange estimate; stat = Wald statistic; pvalue = Wald test p-value; padj = Benjamini-Hochberg adjusted p-value.
- https://cdn.elifesciences.org/articles/81406/elife-81406-fig3-data1-v2.xlsx
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Figure 3—source data 2
Gene ontology terms enriched in genes differentially expressed between fluconazole (FCZ)-treated cells classified in the Ribo-dominant (Rd) cluster 1 (pseudo-bulk samples) compared to FCZ-treated cells in the Stress-dominant (Sd) cluster 4 (pseudo-bulk samples) listed in Figure 3—figure supplement 2B.
- https://cdn.elifesciences.org/articles/81406/elife-81406-fig3-data2-v2.xlsx
Figure 3—figure supplement 1
Differential growth characteristics of strain SC5314 in response to FCZ.
(A) Minimum inhibitory concentration (MIC) of fluconazole (FCZ) determined for strain SC5314. Optical density of cultures grown in liquid YPD for 24 and 48 hr at 30°C in the presence of indicated …
Figure 3—figure supplement 2
Pathway analysis between the Rd and Sd cell clusters.
(A) Empirical cumulative distribution function (ECDF) of signature scores for each cell in the distinct clusters (color) presented in Figure 3B. The y-axis provides the proportion of cells in each …
Figure 4
The Uniform Manifold Approximation and Projection (UMAP) embedding from Figure 3A, which contains fluconazole (FCZ) day 2 and day 3 cells.
Here, each Ribo-dominant (Rd) and Stress-dominant (Sd) cell is colored by its signature score for: (A) C. albicans orthologs of genes upregulated in ribosome assembly stress response (RASTR) and (B) …
Figure 5
Both the Rd and Sd states persist at six days post-fluconazol treatment.
(A) Uniform Manifold Approximation and Projection (UMAP) embedding of fluconazole (FCZ)-treated cells at days 3 and 6 (after resuspension in fresh media at day 3). Leiden clustering identified four …
Appendix 1—figure 1
Fluconazole (FCZ) disk assay results across 6 days.
Two cultures (biological replicates E1 and E2) of C. albicans SC5314 were grown in YPD and spread on three plates (technical replicates R1, R2, and R3). The bright white spot at the center of the …
Appendix 1—figure 2
Diffusion disk assay experiments with fluconazole.
(A) A C. albicans culture with fluconazole (FCZ) disk in the middle of plate. The red radial line represents 1 of 72 measurements taken every 5°. (B) Top is a blown-up and restricted image of the …
Appendix 2—figure 1
Single cell analysies of additional anti-fungal drugs.
(A, B, D–F) Uniform Manifold Approximation and Projection (UMAP) embedding from Figure 3A. Color bar depicts: (A) density of untreated (UT) cells, (B) expression score for the glycolysis signature. …
Appendix 2—figure 2
Differentially expressed genes and pathways associated with comets.
(A) Genes overexpressed in the collection of 19 comets compared to other cells in main clusters (Bayes factor > 2.5 and proportion of non-zero value > 0.2). Color bar indicates the mean expression …
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Appendix 2—figure 2—source data 1
Differentially expressed genes in cells classified in the comet clusters (pseudo-bulk samples) compared to other fluconazole (FCZ)-treated cells (pseudo-bulk samples).
baseMean = the average of the normalized counts taken over all pseudo-bulk samples; log2FoldChange = log2 fold change between the groups; lfcSE = standard error of the log2FoldChange estimate; stat = Wald statistic; pvalue = Wald test p-value; padj = Benjamini-Hochberg adjusted p-value.
- https://cdn.elifesciences.org/articles/81406/elife-81406-app2-fig2-data1-v2.xlsx
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Appendix 2—figure 2—source data 2
Gene ontology terms enriched in genes differentially expressed between comets (pseudo-bulk) and other fluconazole (FCZ)-treated cells (pseudo-bulk) listed in Figure 3—table supplement 1A.
- https://cdn.elifesciences.org/articles/81406/elife-81406-app2-fig2-data2-v2.xlsx
Author response image 1
The heatmap depicts the expression of the most variable IFH1 targets (rows) across cells (columns).
We used MAGIC to impute expression levels (z-score, color bar). Cells are linearly ordered by the overall magnitude of gene expression (mean gene rank). “Clusters” (top) indicate in which cluster …
Author response image 2
Tables
Table 1
(A) Orthologous genes in C. albicans associated with ribosome assembly stress response (RASTR) in S. cerevisiae.
(B) Constitutive Hsf1 target genes.
| RASTR signature (Albert et al., 2019) | ||
|---|---|---|
| DOWN | Ribosome processing | ASC1, RPL10, RPL10A, RPL11, RPL12, RPL13, RPL14, RPL16A, RPL17B, RPL18, RPL19A, RPL2, RPL20B, RPL21A, RPL23A, RPL24A, RPL25, RPL27A, RPL28, RPL30, RPL32, RPL35, RPL37B, RPL38, RPL39, RPL42, RPL5, RPL6, RPL8B, RPL9B, RPP0, RPP1B, RPS1, RPS10, RPS12, RPS13, RPS14B, RPS15, RPS16A, RPS17B, RPS18, RPS19A, RPS20, RPS21, RPS21B, RPS22A, RPS23A, RPS25B, RPS26A, RPS27, RPS27A, RPS3, RPS5, RPS6A, RPS7A, RPS8A, RPS9B, UBI3, YST1 |
| UP | Protein folding, response to heat, proteolysis | UBI4, RPN4, PIN3, STF2, KAR2, MSI3, HSP60, HSP90, HSP70, SIS1, SSA2, HSP104, HSP78, STI1 |
| Glucose and pyruvate metabolic process | CYP1, PMA1, GLK1, TDH3, CDC19, PGK1 | |
| Unknown function | MBF1, KRE30, YDJ1, YBN5, ACT1, UBC4 | |
| Hsf1 constitutive targets (Leach et al., 2016) | ||
| ACE2, ADAEC, AHA1, ALO1, ALS1, ALS3, ALS4, ASR1, BOI2, BUL1, CCP1, CDC37, CDC48, CPR6, CRD2, CTF1, CYC1, CYP1, ERG2, GAP1, GIT3, GLX3, GOR1, GPX2, GPX3, GRP2, HCH1, HSF1, HSP104, HSP21, HSP60, HSP70, HSP78, HSP90, IFA14, ITR1, KAR2, MDJ1, MGE1, MIA40, MNN24, MSI3, PDC11, PGA56, PGA62, POR1, ROB1, RPM2, RPN4, RPS27A, SBA1, SBP1, SGT2, SIS1, SOK1, SSA2, SSC1, STI1, TRX1, TSA1, TSA1B, YDJ1, YWP1, ZCF35, ZPR1, ZWF1 | ||
Appendix 1—table 1
Two biological (independent) C. albicans SC534 cultures and three technical replicates were subjected to fluconazole (FCZ) diffusion disk assay (DDA) experiments and averaged.
The table represents the output from diskimageR over 6 days of imaging. The increase in tolerance, which is measured by the fraction of growth (FoG), from day 1 to day 2 is significant as is the …
| Day | FoG | RAD20 |
|---|---|---|
| 1 | 0.14 | 16.33 |
| 2 | 0.21 | 14.17 |
| 3 | 0.37 | 12.67 |
| 4 | 0.37 | 13.00 |
| 5 | 0.38 | 12.83 |
| 6 | 0.4 | 13.00 |
Additional files
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MDAR checklist
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Appendix 2—figure 2—source data 1
Differentially expressed genes in cells classified in the comet clusters (pseudo-bulk samples) compared to other fluconazole (FCZ)-treated cells (pseudo-bulk samples).
baseMean = the average of the normalized counts taken over all pseudo-bulk samples; log2FoldChange = log2 fold change between the groups; lfcSE = standard error of the log2FoldChange estimate; stat = Wald statistic; pvalue = Wald test p-value; padj = Benjamini-Hochberg adjusted p-value.
- https://cdn.elifesciences.org/articles/81406/elife-81406-app2-fig2-data1-v2.xlsx
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Appendix 2—figure 2—source data 2
Gene ontology terms enriched in genes differentially expressed between comets (pseudo-bulk) and other fluconazole (FCZ)-treated cells (pseudo-bulk) listed in Figure 3—table supplement 1A.
- https://cdn.elifesciences.org/articles/81406/elife-81406-app2-fig2-data2-v2.xlsx
