Monitoring Glycolysis and Respiration Highlights Metabolic Inflexibility of Cryptococcus neoformans
Abstract
:1. Introduction
2. Results
2.1. Assay Optimization for C. neoformans
2.1.1. Assay Medium and Cell Seeding Density
2.1.2. Mitochondrial Stress Test
2.1.3. Glycolytic Rate Test
2.2. Measuring Metabolic Activity of C. neoformans and Human Monocytes in Co-Culture
2.3. Comparing the Metabolic Profiles of C. neoformans WT and the Mutant Strain, Kcs1Δ
2.4. Comparing the Metabolic Profiles of Three Yeast Species
3. Discussion
4. Materials and Methods
4.1. Fungal Strains, Media and Reagents
4.2. Seahorse XFe24 Analyzer Setup and Optimization for Measuring Metabolism in C. neoformans
4.2.1. Day before the Run
Sensor Cartridge Preparation
Fungal Cell Preparation
4.2.2. Day of the Run
CD14 Monocyte Preparation
Fungal Cell Preparation
Sensor Cartridge Preparation
Assay Setup on Seahorse Analyzer
4.3. Co-Culture Assay
- For host-pathogen co-culture samples, 100 µL of 250,000 CD14+ monocytes in Assay medium (with 10 mM glucose) were combined with 500,000 C. neoformans cells. The cells were mixed and plated on XF tissue culture plate pre-coated with Cell-Tak (Corning) following manufacturer’s instruction.
- For cryptococcal control samples, 100 µL of Assay medium were added to cryptococcal cells. The cells were resuspended and plated on XF tissue culture plate.
- For monocyte control samples, 100 µL of 250,000 CD14+ monocytes in Assay medium were plated on XF tissue culture plate.
- The plate was incubated at 37 °C, 5% CO2 for 30 min.
- To improve the attachment and provide even dispersal of the cells, the plate was centrifuged at 100× g for 1 min, deceleration 3. Then the plate was turned 180° and similarly centrifuged.
- 400 µL Assay medium were slowly added to each well.
- The plate was incubated at 37 °C without CO2 enrichment until ready to insert to the Analyzer (~1.5 h from the start of co-incubation).
- Rot/AA 0.5 µM and 2DG 50 mM were injected into the plate wells during the course of the Assay.
4.4. Data Normalization for Fungal Cells
- Process → Sharpen (improves separation between cells)
- Process → Binary → Convert to Mask (creates binary black and white image)
- Select representative area of the micrograph using buttons in the main ImageJ menu, or use the entire image by default
- Analyze → Analyze Particles (select Size as 3-infinity [in pixels]; tick “Summarize” option). If “%Area” is not displayed in the “Summary” window, tick “Area” option in the Analyze → Set Measurements window
4.5. DiOC6 Staining
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Lev, S.; Li, C.; Desmarini, D.; Liuwantara, D.; Sorrell, T.C.; Hawthorne, W.J.; Djordjevic, J.T. Monitoring Glycolysis and Respiration Highlights Metabolic Inflexibility of Cryptococcus neoformans. Pathogens 2020, 9, 684. https://doi.org/10.3390/pathogens9090684
Lev S, Li C, Desmarini D, Liuwantara D, Sorrell TC, Hawthorne WJ, Djordjevic JT. Monitoring Glycolysis and Respiration Highlights Metabolic Inflexibility of Cryptococcus neoformans. Pathogens. 2020; 9(9):684. https://doi.org/10.3390/pathogens9090684
Chicago/Turabian StyleLev, Sophie, Cecilia Li, Desmarini Desmarini, David Liuwantara, Tania C. Sorrell, Wayne J. Hawthorne, and Julianne T. Djordjevic. 2020. "Monitoring Glycolysis and Respiration Highlights Metabolic Inflexibility of Cryptococcus neoformans" Pathogens 9, no. 9: 684. https://doi.org/10.3390/pathogens9090684