This readme.txt file was generated on <2023/12/28> by Recommended citation for the data: Zhou, Xin; Hilk, Audrey; Solis, Norma V; Scott, Nancy; Beach, Annette; Soisangwan, Natthapon; Billings, Clara L; Burrack, Laura S; Filler, Scott G; Selmecki, Anna. (2024). Data supporting “Single-cell detection of copy number changes reveals dynamic mechanisms of adaptation to antifungals in vitro and in vivo”. Retrieved from the Data Repository for the University of Minnesota, https://doi.org/10.13020/pg0z-ag23 ------------------- GENERAL INFORMATION ------------------- 1. Title of Dataset Single-cell detection of copy number changes reveals dynamic mechanisms of adaptation to antifungals in vitro and in vivo 2. Author Information Principal Investigator Contact Information Name: Anna Selmecki Institution: University of Minnesota Address: 3-105 Microbiology Research Facility, 689 SE 23rd Ave, Minneapolis, MN 55455 Email: selmecki@umn.edu ORCID: 0000-0003-3298-2400 Associate or Co-investigator Contact Information Name: Xin Zhou Institution: University of Minnesota Address: 3-151 Microbiology Research Facility, 689 SE 23rd Ave, Minneapolis, MN 55455 Email: zxin@umn.edu ORCID: 0000-0002-5323-173X 3. Date published or finalized for release: 2025_01_01 4. Date of data collection (single date, range, approximate date) 2022/01/04-2023/03/29 5. Geographic location of data collection (where was data collected?): University of Minnesota, 3-153 Microbiology Research Facility University of Minnesota flow cytometry core facility 6. Information about funding sources that supported the collection of the data: National Institutes of Health (R01 AI143689) Burroughs Wellcome Fund Investigator in the Pathogenesis of Infectious Diseases Award (#1020388) 7. Overview of the data (abstract): Genomic copy number changes are associated with antifungal drug resistance and virulence across diverse fungal pathogens. Despite the high prevalence of both gain and loss events, the rate and dynamics of these genomic changes in the presence of antifungal drugs is not known for any organism. We optimized a dual-fluorescent reporter system in the diploid pathogen Candida albicans to quantify copy number variation (CNV) and loss of heterozygosity (LOH) at the single cell level with flow cytometry. We followed the rate and dynamics of CNV and LOH at two distinct genomic locations during parallel evolution experiments in the presence and absence of antifungal drugs in vitro and in a murine model of infection. Copy number changes were rapid and dynamic during adaptation to three different concentrations of the azole drug fluconazole. The fluorescent reporters revealed competing sub-populations with distinct genotypes in the drug-evolved lineages. Extensive whole genome sequencing identified recurrent genotypes that cause increased competitive fitness in the presence of antifungal drug. Specifically, at low concentrations of drug, chromosome 3 (Chr3) LOH frequently arose and led to significantly increased competitive fitness. In contrast, at high drug concentrations, Chr3 and Chr6 aneuploidy arose together, and these concurrent aneuploidies conferred multi-azole tolerance. In the murine model, copy number changes were only detected in isolates recovered from mice treated with fluconazole, and included whole-genome duplication events resulting in polyploidy. This study provides the first quantitative evidence for the incredible speed at which diverse genotypes arise and undergo dynamic population-level fluctuations during adaptation to antifungal drugs in vitro and in vivo. -------------------------- SHARING/ACCESS INFORMATION -------------------------- 1. Licenses/restrictions placed on the data: CC0 1.0 Universal (https://creativecommons.org/publicdomain/zero/1.0/) 2. Links to publications that cite or use the data: To be determined 3. Was data derived from another source? No If yes, list source(s): 4. Terms of Use: Data Repository for the U of Minnesota (DRUM) By using these files, users agree to the Terms of Use. https://conservancy.umn.edu/pages/drum/policies/#terms-of-use --------------------- DATA & FILE OVERVIEW --------------------- 1. File Folder List A. Folder name: Main figures Fig2-4 Short description: Flow cytometry data for figures 2, 3A, 3B, 4B, 4D a. Folder name: Fig2 Short description: Flow cytometry data for all fluconazole evolved lineages b. Folder name: Fig3A Short description: Flow cytometry data from P1-P10 for selected lineages c. Folder name: Fig3B Short description: Competitive assay for GFP-sweep lineages d. Folder name: Fig4B Short description: FACS related flow cytometry data e. Folder name: Fig4D Short description: Competitive assay for FACS sorted LOH cells B. Folder name: Main figures Fig6C Short description: Fluorescent changes of mice evolved single colonies C. Folder name: Main figures Fig6E&7 a. Folder name: Fig6E Short description: Ploidy for mice evolved single colonies b. Folder name: Fig7A Short description: Growth curve for mice evolved single colonies c. Folder name: Fig7B Short description: Competitive assay for high-fitness mice samples D. Folder name: Supplementary figures and tables a. Folder name: FigS2 Short description: Flow cytometry data from P10-P15 for selected lineages b. Folder name: FigS4 Short description: Flow cytometry data for selection of single colonies from Chr3_FLC1_L5 c. Folder name: Table S1_1 Short description: Ploidy for FACS sorted single cells d. Folder name: Table S1_2 Short description: Ploidy for fluconazole evolved strains 2. Relationship between files: Independent files support different figures. File / folder tree: ├── Main figures Fig2-4/ │ ├── Fig2/ │ │ └── Flow cytometry data for all fluconazole evolved lineages │ ├── Fig3A/ │ │ └── Flow cytometry data from P1-P10 for selected lineages │ ├── Fig3B/ │ │ └── Competitive assay for GFP-sweep lineages │ ├── Fig4B/ │ │ └── FACS related flow cytometry data │ └── Fig4D/ │ └── Competitive assay for FACS sorted LOH cells ├── Main figures Fig6C/ │ └── Fluorescent changes of mice evolved single colonies ├── Main figures Fig6E&7/ │ ├── Fig6E/ │ │ └── Ploidy for mice evolved single colonies │ ├── Fig7A/ │ │ └── Growth curve for mice evolved single colonies │ └── Fig7B/ │ └── Competitive assay for high-fitness mice samples ├── Supplementary figures and tables/ │ ├── FigS2/ │ │ └── Flow cytometry data from P10-P15 for selected lineages │ └── FigS4/ │ └── Flow cytometry data for selection of single colonies from Chr3_FLC1_L5 ├── Table S1_1/ │ └── Ploidy for FACS sorted single cells └── Table S1_2/ └── Ploidy for fluconazole evolved strains -------------------------- METHODOLOGICAL INFORMATION -------------------------- 1. Description of methods used for collection/generation of data: These methods include the detection of fluorescent changes, ploidy and growth curve. Flow cytometry sampling and analysis Before flow cytometry analysis, all isolates were inoculated in YPAD from frozen stocks and incubated for 12-16 hrs at 30℃, 220 rpm. Cultures were diluted in PBS and at least 10,000 singlets were gated and analyzed for each sample using a Cytek Aurora flow cytometer (R0021). 405-nm lasers were used to excite the BFP proteins and 458/15 filters were used to detect the BFP emission signals. 488-nm lasers were used to excite the GFP proteins and 508/20 filters were used to detect the GFP emission signals. Data were analyzed using FlowJo (https://www.flowjo.com/solutions/flowjo/downloads) (v10.8.1). The 12 lineages evolved in 0 µg/ml were used as the 1:1 ratio (BFP:GFP) control for each passage, eliminating growth condition effects on fluorescence changes. By using background and mono-fluorescence controls, we removed doublets and drew gates to fit the mono- and dual-fluorescent cells and non-fluorescent/dead cells: BFP+ GFP-, BFP- GFP+, BFP+ GFP+, and BFP- GFP- . BFP+ GFP- and BFP- GFP+ indicated cells that only have BFP (BFP-only) or GFP (GFP-only) fluorescence, respectively. Within the dual-fluorescent gate, BFP/GFP progenitor strains with only one copy of both BFP and GFP were used to generate a 1:1 ratio (BFP vs GFP) gate. The proportions of each subpopulation were calculated and normalized by the total viable cell count and used to generate the stacked plots. Growth curve Isolates were inoculated in 2% dextrose YPAD from frozen stocks and incubated for 16 hrs at 30℃, 220 rpm. Cultures were diluted in fresh 1% dextrose YPAD to a final OD600 of 0.01. 20 µl of this dilution was added to a 96-well NUNC plate containing 180 µl of 1% dextrose YPAD supplemented with or without drug and incubated at 30℃ in a BioTek Epoch 2 microplate spectrophotometer shaking in a double-orbital (237 rpm). OD600 readings were taken every 15 minutes for 48 hrs. Every isolate was conducted in three independent replicates. Ploidy analysis (DNA-PI staining) Cells were prepared as described previously. Isolates were inoculated in 2% dextrose YPAD from frozen stocks and incubated overnight at 30℃, 220 rpm to a cell density of 1x107 cells/ml. Cultures were gently spun down at 3000 rpm for 3 minutes and the supernatant was removed. Cell pellets were resuspended in 70% ethanol, then washed twice with 50 mM sodium citrate. Following washing, cells were spun down and resuspended in 50 mM sodium citrate containing 0.5 mg/ml RNase A. Cells were treated with RNase A at 37℃ for at least two hrs, then stained with 25 µg/ml propidium iodide (PI) at 37℃ in the dark overnight. Samples were diluted in 50 mM sodium citrate, and at least 10,000 singlets were analyzed using a Cytek Aurora flow cytometer (R0021). The 488-nm lasers were used to excite the PI-staining and 618/24 filters were used to detect the PI-staining emission signals. Data were analyzed using FlowJo (https://www.flowjo.com/solutions/flowjo/downloads) (v10.8.1). 2. Methods for processing the data: These are raw flow cytometry data without processing. 3. Instrument- or software-specific information needed to interpret the data: Data were collected on a Cytek Aurora flow cytometer (R0021). Data were analyzed using FlowJo (https://www.flowjo.com/solutions/flowjo/downloads) (v10.8.1) 4. Standards and calibration information, if appropriate: See the description of methods. 5. Environmental/experimental conditions: In-vitro evolution experiment BFP/GFP tagged progenitors AMS5178 (Chr3) and AMS5192 (Chr5) were plated on YPAD agar and incubated for 24 hrs at 30℃. Twelve single colonies from each strain were selected and inoculated in liquid YPAD in deep-well 96-well plates. Plates were sealed with BreathEASIER (Electron Microscope Sciences) tape and cultured for 16-18 hrs at 30℃, 220 rpm. From the overnight culture, a 1:1000 dilution was made into four culture conditions: (i) YPAD with 0 µg/ml FLC, (ii) YPAD with 1 µg/ml FLC, (iii) YPAD with 4 µg/ml FLC, and (iv) YPAD with 8 µg/µml FLC in deep-well 96-well plates. Cells were cultured at 30℃, 220 rpm, and at each 48-hour timepoint, cells were resuspended and transferred via 1:1000 dilution to fresh YPAD medium containing the designated concentration of fluconazole. After ten total transfers (P1-P10), FLC was removed from culture conditions. Transfers for all lineages continued for another 5 transfers (P11-P15) in YPAD culture conditions, with 1:1000 dilutions every 48 hrs (Fig S1A). At each transfer, cells were collected for storage at -80℃. Mouse model of Candidiasis C. albicans progenitor strains AMS5178 (Chr3) and AMS5192 (Chr5) were inoculated in YPAD and incubated for 16 hrs at 30℃. On day 0, immunocompetent ICR mice were injected with 4x104 (AMS5178) or 1x105 (AMS5192) cells. Starting on day 1, mice were administered FLC at 0.5, 2, or 5 mg/kg by gavage. Control mice received PBS. The mice were weighed every day to determine the FLC dose. Drug treatment continued for 5 days. On day 6, mice were sacrificed and tissues including kidney, brain, and liver were harvested. These tissues were weighed, homogenized, and quantitatively cultured for CFU counting and fungal cell recovery. 6. Describe any quality-assurance procedures performed on the data: Quality control was performed for the Cytek Aurora flow cytometer (R0021) daily using SpectroFlo QC Beads. 7. People involved with sample collection, processing, analysis and/or submission: Xin Zhou, Anna Selmecki, Audrey Hilk ----------------------------------------- DATA-SPECIFIC INFORMATION FOR: Folder Fig2 ----------------------------------------- Flow cytometry data for all fluconazole evolved lineages layout: A1-A12: Lineage1-Lineage 12 in YPAD E1-E12: Lineage1-Lineage 12 in YPAD +1µg/ml FLC C1-C12: Lineage1-Lineage 12 in YPAD +4µg/ml FLC G1-G12: Lineage1-Lineage 12 in YPAD +8µg/ml FLC File was named by the experiment, progenitor, passage and sample ID. ----------------------------------------- DATA-SPECIFIC INFORMATION FOR: Folder Fig3A ----------------------------------------- Flow cytometry data from P1-P10 for selected lineages Chr3: A2-J2: P1-P10 Chr3_FLC1_L4 A3-J3: P1-P10 Chr3_FLC1_L5 A4-J4: P1-P10 Chr3_FLC1_L9 A6-J6: P1-P10 Chr3_FLC4_L8 A7-J7: P1-P10 Chr3_FLC4_L9 A9-J9: P1-P10 Chr3_FLC8_L10 Chr5: A1, A7, D1, D7, E1, E7, F1, F7, G1, G7: P1-P10 Chr5_FLC1_L6 A3, A9, D3, D9, E3, E9, F3, F9, G3, G9: P1-P10 Chr5_FLC4_L1 A4, A10, D4, D10, E4, E10, F4, F10, G4: G10: P0-P10 Chr5_FLC8_L2 A5, A11, D5, D11, E5, E11, F5, F11, G5, G11: P1-P10 Chr5_FLC8_L6 File was named by the experiment, progenitor, and sample ID. ----------------------------------------- DATA-SPECIFIC INFORMATION FOR: Folder Fig3B ----------------------------------------- Competitive assay for GFP-sweep lineages 24hr in YPAD: B4+H1: Chr3_FLC1_L4 vs WT control B8+H2: Chr3_FLC1_L9 vs WT control B12+H3: Chr5_FLC8_L2 vs WT control 24hr in YPAD+1µg/ml: D4+H5: Chr3_FLC1_L4 vs WT control D8+H6: Chr3_FLC1_L9 vs WT control D12+H7: Chr5_FLC8_L2 vs WT control 24hr in YPAD+4µg/ml: F4+H9: Chr3_FLC1_L4 vs WT control F8+H10: Chr3_FLC1_L9 vs WT control F12+H11: Chr5_FLC8_L2 vs WT control ----------------------------------------- DATA-SPECIFIC INFORMATION FOR: Folder Fig4B ----------------------------------------- FACS related flow cytometry data AMS5178: Chr3 BFP/GFP AMS5192: Chr5 BFP/GFP LOH rates: .fcs used to determine thee rates of BFP-only or GFP-only cells FACS sorted single cells: flow cytometry data for all sorted single cells Recovery from sorted single cells: flow cytometry data for 36hr culture of sorted single cells ----------------------------------------- DATA-SPECIFIC INFORMATION FOR: Folder Fig4D ----------------------------------------- 24hr in YPAD D1-D3: Chr3 segmental LOH AA(BFP) vs Ch3 segmental LOH BB(GFP) D4-D6: Chr3 whole Chr LOH AA(BFP) vs Chr3 whole Chr LOH BB(GFP) D7-D9: Chr5 whole Chr LOH AA(GFP) vs Ch5 whole Chr LOH BB(BFP) D10-D12: Chr5 segmental LOH AA(GFP) vs Ch5 segmental LOH BB(BFP) 24hr in YPAD+1µg/ml FLC H1-H3: Chr3 segmental LOH AA(BFP) vs Ch3 segmental LOH BB(GFP) H4-H6: Chr3 whole Chr LOH AA(BFP) vs Chr3 whole Chr LOH BB(GFP) H7-H9: Chr5 whole Chr LOH AA(GFP) vs Ch5 whole Chr LOH BB(BFP) H10-D12: Chr5 segmental LOH AA(GFP) vs Ch5 segmental LOH BB(BFP) ----------------------------------------- DATA-SPECIFIC INFORMATION FOR: Folder Fig6C ----------------------------------------- Fluorescent changes of mice evolved single colonies AMS5178: Chr3 BFP/GFP AMS5192: Chr5 BFP/GFP File was named by the date that flow cytometry was performed, progenitor, mouse number, and treatment (PBS or FLC). ----------------------------------------- DATA-SPECIFIC INFORMATION FOR: Folder Fig6E ----------------------------------------- Ploidy for mice evolved single colonies AMS5178: Chr3 BFP/GFP AMS5192: Chr5 BFP/GFP File was named by the date that flow cytometry was performed, progenitor, mouse number, and treatment (PBS or FLC). ----------------------------------------- DATA-SPECIFIC INFORMATION FOR: Folder Fig7A ----------------------------------------- -----Info about each spreadsheet here----- File: 2022_10_18 5178_Mouse2_FLC2 48hr growth curve.xls 48hr growth curve raw OD600 for fungal cells recovered from AMS5178 (Chr3 BFP/GFP progenitor) infected mouse 2 which were treated with 2mg/kg FLC. Each column represents an independent sample A1-H12. Plate1&2: YPAD Plate3&4: YPAD+1µg/ml FLC File: 2022_10_20 5178_Mice1-5_FLC5_48hr growth curve.xls 48hr growth curve raw OD600 for fungal cells recovered from AMS5178 (Chr3 BFP/GFP progenitor) infected mice1-5 which were treated with 5mg/kg FLC. Each column represents an independent sample A1-H12. Plate1&2: YPAD Plate3&4: YPAD+1µg/ml FLC File: 2022_10_25 5178_Mouse1_PBS_48hr growth curve.xls 48hr growth curve raw OD600 for fungal cells recovered from AMS5178 (Chr3 BFP/GFP progenitor) infected mouse1 which were treated with PBS. Each column represents an independent sample A1-H12. Plate1&2: YPAD Plate3&4: YPAD+1µg/ml FLC File: 2022_11_10 5192_Mouse2-5_FLC2_48hr growth curve.xls 48hr growth curve raw OD600 for fungal cells recovered from AMS5192 (Chr5 BFP/GFP progenitor) infected mouse2-5 which were treated with 2mg/kg FLC. Each column represents an independent sample A1-H12. Plate1&2: YPAD Plate3&4: YPAD+1µg/ml FLC File: 2022_11_12 5192_Mouse4+5_FLC5_48hr growth curve.xls 48hr growth curve raw OD600 for fungal cells recovered from AMS5192 (Chr5 BFP/GFP progenitor) infected mouse 4 and 5 which were treated with 5mg/kg FLC. Each column represents an independent sample A1-H12. Plate1&2: YPAD Plate3&4: YPAD+1µg/ml FLC Folder: AMS5178_FLC0.5_mouse2 48hr growth curve raw OD600 for fungal cells recovered from AMS5178 (Chr3 BFP/GFP progenitor) infected mouse2 which were treated with 0.5mg/kg FLC. Each column represents an independent sample A1-H12. File: 2023_05_11 5178_FLC0.5_mouse2 48hr GC FLC1of2.xls File: 2023_05_11 5178_FLC0.5_mouse2 48hr GC FLC2of2.xls File: 2023_05_11 5178_FLC0.5_mouse2 48hr GC YPAD.xls Folder: AMS5192_FLC0.5_mouse1 48hr growth curve raw OD600 for fungal cells recovered from AMS5192 (Chr5 BFP/GFP progenitor) infected mouse1 which were treated with 0.5mg/kg FLC. Each column represents an independent sample A1-H12. File: 2023_04_05 5192_FLC0.5_mouse1 48hr GC FLC1.xls File: 2023_04_05 5192_FLC0.5_mouse1 48hr GC FLC2.xls File: 2023_04_05 5192_FLC0.5_mouse1 48hr GC YPAD.xls ----------------------------------------- DATA-SPECIFIC INFORMATION FOR: Folder Fig7B ----------------------------------------- Competitive assay for high-fitness mice samples 24hr in YPAD+1µg/ml FLC: A1-A3: WT control vs E9 A4-A6: WT control vs E10 A7-A9: WT control vs E12 A10-A12: WT control vs F6 24hr in YPAD: B1-B3: WT control vs E9 B4-B6: WT control vs E10 B7-B9: WT control vs E12 B10-B12: WT control vs F6 ----------------------------------------- DATA-SPECIFIC INFORMATION FOR: Folder FigS2 ----------------------------------------- Flow cytometry data from P10-P15 for selected lineages Chr3 C8: Chr3_FLC4_L8 Chr3 C9: Chr3_FLC4_L9 Chr3 E5: Chr3_FLC1_L5 Chr3 G10: Chr3_FLC4_L10 Chr5 E6: Chr5_FLC1_L6 Chr5 E7: Chr5_FLC1_L7 Chr5 E8: Chr5_FLC1_L8 Chr5 G6: Chr5_FLC8_L6 ----------------------------------------- DATA-SPECIFIC INFORMATION FOR: Folder FigS4 ----------------------------------------- Flow cytometry data for selection of single colonies from Chr3_FLC1_L5 S1-S12: Chr3_FLC1_L5 Single colony 1-12 ----------------------------------------- DATA-SPECIFIC INFORMATION FOR: Folder Table S1_1 ----------------------------------------- Ploidy for FACS sorted single cells 1 2 3 4 5 6 7 8 9 A A3 A4 A5 B5 B6 B10 C4 C5 C7 B D10 E5 E12 F2 F11 G1 G3 H7 H9 C A1 A8 B5 B8 B10 B11 B12 C5 C6 D D5 D11 E4 E7 E8 F2 G3 G9 G12 E A2 A3 A4 B5 B7 B10 C4 C7 C12 F E5 E7 E8 F1 F7 F8 G1 G5 G11 G A1 A2 A3 A5 A6 A8 A9 B6 B9 H D3 D4 D5 D8 D10 D11 D12 E2 E6 A-B: Chr3 BFP-only sorted single colonies C-D: Chr3 GFP-only sorted single colonies E-F: Chr5 BFP-only sorted single colonies G-H: Chr5 GFP-only sorted single colonies ----------------------------------------- DATA-SPECIFIC INFORMATION FOR: Folder Table S1_2 ----------------------------------------- Ploidy for fluconazole evolved strains File name by experiment, progenitor, passage and sample name.