Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Summary

Here, we present a stepwise protocol to investigate the mitochondrial respiration and glycolytic function in Candida Albicans using an extra flux analyzer.

Abstract

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular respiration, oxidative metabolism, signal transduction, and apoptosis. Consequently, mitochondrial dysfunction is reported to play an important role in the antifungal drug tolerance and virulence of pathogenic fungi. Recent data have also led to the recognition of the importance of the mitochondria as an important contributor to fungal pathogenesis. Despite the importance of the mitochondria in fungal biology, standardized methods to understand its function are poorly developed. Here, we present a procedure to study the basal oxygen consumption rate (OCR), a measure of mitochondrial respiration, and extracellular acidification rates (ECAR), a measure of glycolytic function in C. albicans strains. The method described herein can be applied to any Candidaspp. strains without the need to purify mitochondria from the intact fungal cells. Furthermore, this protocol can also be customized to screen for inhibitors of mitochondrial function in C. albicans strains.

Introduction

Invasive fungal infections kill over 1.5 million people a year worldwide. This number is on the rise due to an increase in the numbers of people living with compromised immunity, including the elderly, premature infants, transplant recipients, and cancer patients¹. C. albicans is an opportunistic human fungal pathogen that is a part of the human microflora. It also inhabits mucosal surfaces and the gastrointestinal tract as a commensal organism. C. albicans produces serious systemic disease in people who have immune deficiencies, who have undergone surgery, or who have been treated with long courses of antibiotics. The Candida species rank among the top three to four causes of nosocomial infectious diseases (NID) in humans^2,3,4,5,6,7. The annual global number of Candida bloodstream infections is estimated to be ~400,000 cases, with associated mortalities of 46-75%¹. The annual mortality because of candidiasis is roughly 10,000 in the United States alone. The extent of NID caused by fungi is also reflected in astronomical patient expenses⁵. In the United States, the yearly expense for the treatment of invasive fungal infections surpasses $2 billion, adding a huge strain to already overburdened health care system. Currently, available standard antifungal therapies are limited because of toxicity, increasingly prevalent drug resistance, and drug-drug interactions. Therefore, there is an urgent need to identify new antifungal drug targets that will result in better treatment options for high-risk patients. However, the discovery of new drugs acting on fungal targets is complicated because fungi are eukaryotes. This greatly limits the number of fungal-specific drug targets.

Recent studies have indicated that mitochondria are a critical contributor to the fungal virulence and tolerance to antifungal drugs since mitochondria are important for cellular respiration, oxidative metabolism, signal transduction, and apoptosis^8,9,10,11. Both glycolytic and non-glycolytic metabolism are essential for the survival of C. albicans in the mammalian host^{12,13,14,15,16}. Furthermore, several C. albicans mutants lacking mitochondrial proteins, such as Goa1, Srr1, Gem1, Sam37 etc. have been shown to be defective in filamentation, an important virulence factor of C. albicans^{17,18,19,20,21,22}. In addition, these mutants were also shown to be attenuated for virulence in a mouse model of disseminated candidiasis^{17,18,19,20,21,22}. Thus, fungal mitochondria represent an attractive target for drug discovery. However, the study of mitochondrial function in C. albicans is challenging because C. albicans is petite negative²³, which means that it cannot survive without the mitochondrial genome.

Here, we describe a protocol that can be used to investigate mitochondrial and glycolytic function in C. albicans without the need to purify mitochondria. This method can also be optimized to investigate the effect of the genetic manipulation or chemical modulators on mitochondrial and glycolytic pathways in C. albicans.

Protocol

NOTE: The detailed stepwise protocol of the assay is described below, and the schematic protocol is shown in Figure 1.

1. C. albicans strains and growth conditions

1. Grow the C. albicans strains in liquid Yeast Extract-Peptone-Dextrose (YPD) medium at 30 °C in an incubator shaker overnight.
   NOTE: Maintain Candida strains as frozen stocks and grow on YPD agar (1 % yeast extract, 2% peptone, 2% dextrose, and 2% agar).

2. Preparation of reagents

1. Prepare the assay medium as follows:
   1. For mitochondrial function assay, dissolve 1.04 g Roswell Park Memorial Institute (RPMI) 1640 powder and 2 g glucose (2%) in 90 mL sterile water and warm the media to 37 °C. Adjust the pH to 7.4 using 5 M NaOH and make up the volume to 100 mL with sterile water.
   2. For glycolytic stress assay, dissolve 1.04 g RPMI 1640 powder alone in 90 mL sterile water, warm the media to 37 °C and adjust the pH to 7.4. Make up the volume to 100 mL with sterile water. RPMI 1640 powder has no bicarbonate, which is critical to monitor the pH change as a measure of glycolysis during the assay.
2. Injection compounds.
   1. Prepare 1 M glucose stock in sterile water and store at -20 °C.
   2. Prepare 100 mM oligomycin stock in dimethyl sulfoxide (DMSO), aliquot in small volumes and store at -20 °C.
   3. Prepare 100 mM antimycin A stock in DMSO, aliquot in small volumes and store at -20 °C.
   4. Prepare 100 mM SHAM (salicylhydroxamic acid) stock in ethanol on the day of assay.
   5. Prepare 1 M KCN in the sterile water on the day of the assay.

3. Coating of the assay plate with Poly-D-Lysine (PDL)

NOTE: Perform all the below steps in a laminar hood.

1. Dissolve Poly-D Lysine in tissue culture grade water to make 50 µg/mL final concentration. Mix it well and aliquot into a 1.5 mL microcentrifuge tubes and store at -20 °C for the long term.
   NOTE: 50 µL per well is needed, and for 24 wells, 1.2 mL is required. Therefore, aliquot at least 1.3 mL per microcentrifuge tube.
2. Add 50 µL per well and incubate at room temperature with the lid covered for 1-2 h.
3. Aspirate the solution and rinse one time with 500 µL sterile tissue culture grade water.
4. Open the lid and allow the wells to air dry. Use the plate on the same day or store at 4 °C for a maximum of 2-3 days.

4. Hydration of sensor cartridge

NOTE: Perform this step one day before the experiment.

1. Open the extra flux Assay Kit and remove the contents. Place the sensor cartridge upside down next to the utility plate (Figure 2).
2. Fill each well of the utility plate with 1 mL of calibrant and place the sensor cartridge back. Make sure the sensors which contain fluorophores (to measure the oxygen and pH) are submerged in the calibrant.
3. Incubate the sensor cartridge overnight in a non-CO₂ incubator at 37 °C.

5. Growing and seeding cells in the PDL-coated plates

1. Inoculate C. albicans in the YPD broth and grow overnight at 30 °C in a shaker at 200 rpm.
   NOTE: Based on the assay design and the interest, C. albicans can also be grown in YPG or minimal medium.
2. On the day of assay, dilute an appropriate number of cells in the assay medium to yield a final concentration of 100,000 cells per 100 µL.
3. Add 100 µL of the diluted cells into each well of the assay plate except wells A1, B4, C3, and D6, in which add only 100 µL of the assay medium for background correction (Figure 3).
4. Transfer the plate to a non-CO₂ incubator at 37 °C and incubate for 60 min, which will let the cells adhere to the plate surface.

6. Assay Protocol

NOTE: The protocol outlined here is for the 24-well format of the instrument. Volumes will need to be adjusted if another format is used.

1. Mitochondrial function assay
   1. Preparation of compounds
      1. Prepare compounds at 10x concentration for mitochondrial function assay: Prepare 20 mM SHAM, 100 µM Oligomycin, 100 mM KCN, and 20 µM Antimycin A in the corresponding assay medium.
      2. Add 50 µL SHAM into the port A, 55 µL Oligomycin into port B, 62 µL KCN into port C and 68 µL Antimycin A into port D (Figure 4).
2. Glycolytic stress assay
   1. Preparation of compounds
      1. Prepare compounds at 10x concentration for glycolytic stress assay. Prepare 100 mM glucose, 100 µM Oligomycin, 500 mM 2-Deoxy Glucose (2DG) and 20 µM Antimycin A in the corresponding assay medium.
      2. Add 50 µL glucose into port A, 55 µL Oligomycin into port B, 62 µL 2-DG into port C and 68 µL Antimycin A into port D.
3. Employ extra flux analyzer, which measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) of live cells in a 24-well plate format. Set up the assay protocol in advance.
4. Open the extra flux analyzer and set up the assay template by using the assay wizard tab and follow step by step instruction to fill out all the information that pops out during the setup. Generate the group layout as similar to shown in Figure 5. Set up the protocol as shown in Table 1. Set up these layouts well ahead before assay and save in the computer. At the time of assay, restore the saved protocol by opening the corresponding file in the open file option in the assay wizard tab (Figure 5).
5. Load the 10x compounds in the respective ports of the hydrated sensor cartridge containing the calibrant and load in the carrier tray of the extra flux analyzer. Start the calibration by pressing the Start button on the screen.
6. Add 350 µL of the assay medium gently to the cell plate along the side of the wells to minimize cell disturbance to bring the final volume to 450 µL.
7. Replace the utility plate containing the calibrant with the assay plate and continue.
8. Remove the sensor cartridge and the plate once the assay is completed. Save the file in the appropriate destination folder.

7. Data Analysis

1. Use the area under the curve- analysis of variance (AUC-ANOVA) analysis tab in the software to calculate the significant difference between the groups by selecting the respective parameters (OCR or ECAR) as shown in Figure 6.
2. Select the groups that need to be compared.
3. Add to the analysis groups for ANOVA and click Ok.
   NOTE: This AUC ANOVA analysis will add a new sheet to the file in which the AUC is calculated for each group and compared between them by ANOVA. This will give a table of p values to show the significance.

Results

The focus of this protocol is to determine the bioenergetic functions of C. albicans assessed by extra flux analyzer. A C. albicans mutant lacking mitochondrial protein Mam33 is also included along with its complement strain, mam33Δ/Δ::MAM33 to study the effects of the deletion of a mitochondrial protein on OCR and ECAR. MAM33 encodes for a putative mitochondrial acidic matrix protein and its function in Candida is not known.

Discussion

The bioenergetics extra flux assay serves as an excellent tool to read out the mitochondrial function by measuring oxidative phosphorylation (OXPHOS)-dependent oxygen consumption in real-time. In addition, a glycolytic function which is measured as an extracellular acidification rate (change in extracellular pH) can also be investigated at the same time in real-time analysis.

Successful plating of C. albicans in the assay plate is one of the critical steps in the assay because the inc...

Materials

Name	Company	Catalog Number	Comments
RPMI 1640	Corning	MT50020PB
Antimycin A	Sigma	A8674
KCN
Mito stress kit	Agilent	103015-100
Oligomycin	Calbiochem	495455
pH meter	Accumet	AR20
Phenol red	Sigma	P5530
Poly-D lysine	Sigma	P6407
Rotenone	Santa cruz	203242
Seahorse XF24 FluxPak	Agilent	100850-001
SHAM
Sodium Chloride	Amresco	241
Sodium hydroxie pellets	J.T Baker	3722
Tissue culture grade water	Gibco	1523-0147
XF assay calibrant solution	Agilent	100840-000
Yeast extract Peptone Dextrose	Fisher scientific,	BP2469
Yeast extract Peptone Dextrose Agar	Sigma	A1296
Yeast extract Peptone Glycerol	Sigma	G2025