Name: bio-energetics investigation of candida albicans using real-time extracellular flux analysis
Uploaded: 2019-03-19T13:30:00.000+00:00
Duration: 8 min 48 s
Description: Watch this Scientific Journal Video about Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis at JoVE.com

Research in NC lab is supported by a National Institutes of Health (NIH) grant R01AI24499 and a New Jersey Health Foundation (NJHF) grant, #PC40-18.

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
<ol>
	<li>Grow the C. albicans strains&#160;in liquid Yeast Extract-Peptone-Dextrose (YPD) medium at 30 &#176;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).</li>
</ol>
2. Preparation of reagents
<ol>
	<li>Prepare the assay medium as follows:
	<ol>
		<li>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 &#176;C. Adjust the pH to 7.4 using 5 M NaOH and make up the volume to 100 mL with sterile water.</li>
		<li>For glycolytic stress assay, dissolve 1.04 g RPMI 1640 powder alone in 90 mL sterile water, warm the media to 37 &#176;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.</li>
	</ol>
	</li>
	<li>Injection compounds.
	<ol>
		<li>Prepare 1 M glucose stock in sterile water and store at -20 &#176;C.</li>
		<li>Prepare 100 mM oligomycin stock in dimethyl sulfoxide (DMSO), aliquot in small volumes and store at -20 &#176;C.</li>
		<li>Prepare 100 mM antimycin A stock in DMSO, aliquot in small volumes and store at -20 &#176;C.</li>
		<li>Prepare 100 mM SHAM (salicylhydroxamic acid) stock in ethanol on the day of assay.</li>
		<li>Prepare 1 M KCN in the sterile water on the day of the assay.</li>
	</ol>
	</li>
</ol>
3. Coating of the assay plate with Poly-D-Lysine (PDL)
NOTE: Perform all the below steps in a laminar hood.
<ol>
	<li>Dissolve Poly-D Lysine in tissue culture grade water to make 50 &#181;g/mL final concentration. Mix it well and aliquot into a 1.5 mL microcentrifuge tubes and store at -20 &#176;C for the long term. 
	NOTE: 50 &#181;L per well is needed, and for 24 wells, 1.2 mL is required. Therefore, aliquot at least 1.3 mL per microcentrifuge tube.</li>
	<li>Add 50 &#181;L per well and incubate at room temperature with the lid covered for 1-2 h.</li>
	<li>Aspirate the solution and rinse one time with 500 &#181;L sterile tissue culture grade water.</li>
	<li>Open the lid and allow the wells to air dry. Use the plate on the same day or store at 4 &#176;C for a maximum of 2-3 days.</li>
</ol>
4. Hydration of sensor cartridge
NOTE: Perform this step one day before the experiment.
<ol>
	<li>Open the extra flux Assay Kit and remove the contents. Place the sensor cartridge upside down next to the utility plate (Figure 2).</li>
	<li>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.</li>
	<li>Incubate the sensor cartridge overnight in a non-CO2 incubator at 37 &#176;C.</li>
</ol>
5. Growing and seeding cells in the PDL-coated plates
<ol>
	<li>Inoculate C. albicans in the YPD broth and grow overnight at 30 &#176;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.</li>
	<li>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 &#181;L.</li>
	<li>Add 100 &#181;L of the diluted cells into each well of the assay plate except wells A1, B4, C3, and D6, in which add only 100 &#181;L of the assay medium for background correction (Figure 3).</li>
	<li>Transfer the plate to a non-CO2 incubator at 37 &#176;C and incubate for 60 min, which will let the cells adhere to the plate surface.</li>
</ol>
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.
<ol>
	<li>Mitochondrial function assay
	<ol>
		<li>Preparation of compounds
		<ol>
			<li>Prepare compounds at 10x concentration for mitochondrial function assay: Prepare 20 mM SHAM, 100 &#181;M Oligomycin, 100 mM KCN, and 20 &#181;M Antimycin A in the corresponding assay medium.</li>
			<li>Add 50 &#181;L SHAM into the port A, 55 &#181;L Oligomycin into port B, 62 &#181;L KCN into port C and 68 &#181;L Antimycin A into port D (Figure 4).</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Glycolytic stress assay
	<ol>
		<li>Preparation of compounds
		<ol>
			<li>Prepare compounds at 10x concentration for glycolytic stress assay. Prepare 100 mM glucose, 100 &#181;M Oligomycin, 500 mM 2-Deoxy Glucose (2DG) and 20 &#181;M Antimycin A in the corresponding assay medium.</li>
			<li>Add 50 &#181;L glucose into port A, 55 &#181;L Oligomycin into port B, 62 &#181;L 2-DG into port C and 68 &#181;L Antimycin A into port D.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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).</li>
	<li>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.</li>
	<li>Add 350 &#181;L 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 &#181;L.</li>
	<li>Replace the utility plate containing the calibrant with the assay plate and continue.</li>
	<li>Remove the sensor cartridge and the plate once the assay is completed. Save the file in the appropriate destination folder.</li>
</ol>
7. Data Analysis
<ol>
	<li>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.</li>
	<li>Select the groups that need to be compared.</li>
	<li>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.</li>
</ol>

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The authors have nothing to disclose

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 incubation of the cells in the PDL coated plates allow the cells to adhere to the assay plate. Visualization of adhering C. albicans cells is accomplished by light microscopy. Improper cell adherence may lead to cells floating in the wells, which will affect the assay outcome with inconsistent readings between replicates. Similarly, it is important to perform the cell number titration as the first step in this assay to obtain an OCR between 100-300 pmol/min, which is an optimal range. Also, titrating the compounds to obtain the optimal concentration is also critical to achieving the optimal response. This may be performed whenever there is a change in the basal medium composition.
DMEM is the most commonly used basal medium in the extra flux assay for mammalian cells. However, in our hands, RPMI 1640 medium gave the optimal results with C. albicans. This was primarily due to the efficacy of mitochondrial electron transport chain complex inhibitors that worked better in RPMI 1640 medium compared to DMEM.
In this assay, wild type, mam33&#916;/&#916;, and mam33&#916;/&#916;::MAM33 strains of C. albicans were used to assess the mitochondrial respiration and glycolytic efficiency. The results clearly show that the deletion of MAM33 gene reduced the ability of the C. albicans to shift to glycolysis under stress conditions such as inhibition of C-IV (Figure 7C and 7E). Similarly, when cells were subjected to stress condition like glucose starvation, both mitochondrial respiration and glycolytic efficiency is significantly compromised in the mam33&#916;/&#916; mutant but not in the wild type and complement strains (Figure 8). Therefore, depending on experiments, carbon sources in the RPMI 1640 may also be modified to glycerol or galactose, which forces the cells to use OXPHOS rather than glycolysis.
The significance of using the extra flux assay is that by using various substrates and compounds that perturb mitochondrial function, the cells ability to utilize OXPHOS versus glycolysis can be inferred. This may be used as an informative tool to understand the effect of any treatments or gene on the mitochondrial function and glycolysis in Candida albicans without isolating mitochondria. The assay measures basal oxygen consumption rate (OCR), which is a measure of mitochondrial respiration, and extracellular acidification rate (ECAR), which is a measure of glycolytic function in both the presence of glucose and after glucose starvation. The extra flux cellular assay is well employed in a variety of mammalian models26. Despite the medical importance of C. albicans, research into fungal mitochondria has been somewhat hampered due to the lack of standardized experimental procedures to study mitochondrial function and glycolysis in real time.
This method provides an easy and convenient way of investigating the mitochondrial respiration and glycolytic efficiency by using real-time extracellular flux assay in C. albicans. In future, this assay may also be applied to study the drug-efficacy testing using commonly employed antifungals such as fluconazole. Additionally, another potential future application of this method is in drug discovery, especially for screening compounds targeting mitochondrial function of C. albicans.

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 humans2,3,4,5,6,7. 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 apoptosis8,9,10,11. Both glycolytic and non-glycolytic metabolism are essential for the survival of C. albicans in the mammalian host12,13,14,15,16. 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. albicans17,18,19,20,21,22. In addition, these mutants were also shown to be attenuated for virulence in a mouse model of disseminated candidiasis17,18,19,20,21,22. 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.

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

bio-energetics investigation of candida albicans using real-time extracellular flux analysis

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.

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, mam33&#916;/&#916;::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.
Cell numbers for extra flux assay 
We employed 1x 105&#160;C. albicans wild type cells per well in the extra flux assay, which showed an OCR of 145 pmol/min (Figure 7), which is within the optimal range of 100-300 pmol/min for any extracellular flux assay. It is also important to titrate the cell number to obtain an optimal OCR before the assay or when there is a change in growth medium composition between different assays.
Analysis of data 
The area under the curve- analysis of variance (AUC-ANOVA) analysis tab in the software was used to calculate the significant difference between the groups by selecting the respective parameters (OCR or ECAR) as shown in Figure 7. This analysis added a new sheet to the file in which the AUC was calculated for each group and compared between them by ANOVA automatically, which gave a table of p values.
Mitochondrial function assay 
In the mitochondrial stress test, the assay starts with measuring the basal oxygen consumption rate (OCR) followed by injection of inhibitors of an alternate oxidase (AOX), ATP synthase, and electron transport chain complexes, IV and III. In addition to the classical respiratory chain, C. albicans also utilizes the AOX pathway for energy generation24. Therefore, we investigated the effect of AOX system on OCR by inhibiting its activity by using salicylhydroxamic acid (SHAM)25. The mitochondrial function, which was measured as an oxygen consumption rate (OCR) in pmol/min at basal and after injecting respective inhibitors is shown in Figure 7. The basal OCR of wild type, mam33&#916;/&#916;, and mam33&#916;/&#916;::MAM33 strains showed no differences (Figure 7A and B). Similarly, no significant difference in the basal ECAR was observed between these strains (Figure 7C and D). However, by inhibiting complex IV by KCN, wild type and mam33&#916;/&#916;::MAM33 showed a substantial shift toward the glycolysis in contrast to mam33&#916;/&#916;, which significantly failed to show a compensatory glycolytic shift (Figure 7E), suggesting that C-IV dependent glycolytic pathway is impaired in the mam33&#916;/&#916; mutant compared to wild type and mam33&#916;/&#916;::MAM33 strains.
Glycolytic stress assay 
In the glycolytic stress test, cells were starved for glucose for 1 h and the basal OCR and ECAR was measured. Upon starvation, mam33&#916;/&#916; showed significantly lower OCR (Figure 8A and B) and ECAR (Figure 8C and D) compared to the wild type and mam33&#916;/&#916;::MAM33 strains suggesting an impaired utilization of glucose for both respiration and glycolysis when cells are forced to the starvation condition. After injecting glucose, all strains showed stimulation of both OCR and ECAR. Although oligomycin has less effect on both OCR and ECAR, 2-DG, which is a competitive inhibitor of glycolysis, inhibits both OCR and ECAR. Specifically, 2-DG partially inhibits OCR, which is further inhibited by Antimycin A. In contrast, 2-DG almost completely inhibits ECAR.
<img alt="Table 1" src="/files/ftp_upload/58913/58913table1.jpg" /> 
Table 1. Protocol commands for the assay
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58913/58913fig1.jpg" /> 
Figure 1. Schematic representation of the experiment <a href="https://www.jove.com/files/ftp_upload/58913/58913fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58913/58913fig2.jpg" /> 
Figure 2. Sensor cartridge (green) placed upside down near the utility plate before adding the calibrant. Fluorophores are indicated by arrows. <a href="https://www.jove.com/files/ftp_upload/58913/58913fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58913/58913fig3.jpg" /> 
Figure 3. Cell culture plate showing background wells A1, B4, C3, and D6. The blue mark shows the notch, oriented to the lower left. <a href="https://www.jove.com/files/ftp_upload/58913/58913fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58913/58913fig4.jpg" /> 
Figure 4. The top portion of the sensor cartridge showing ports A, B, C and D. The arrow mark shows the notch, oriented to the lower left. <a href="https://www.jove.com/files/ftp_upload/58913/58913fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58913/58913fig5.jpg" /> 
Figure 5. The layout of wells and group assignment as visualized in the software. WT- wild type; MT- mam33&#916;/&#916; mutant; COMP- mam33&#916;/&#916;::MAM33 complement strain. Arrow mark shows the open file tab. <a href="https://www.jove.com/files/ftp_upload/58913/58913fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58913/58913fig6.jpg" /> 
Figure 6. The area under the curve (AUC) ANOVA analysis tab to calculate the significance between the groups. Select the groups that need to be compared and add to the analysis groups for ANOVA and click ok. 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. <a href="https://www.jove.com/files/ftp_upload/58913/58913fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58913/58913fig7.jpg" /> 
Figure 7.&#160;Mitochondrial function assay in C. albicans. Oxygen consumption and extracellular acidification rates were measured in wild type, mam33 &#916;/&#916;, and mam33 &#916;/&#916;::MAM33 strains of C. albicans. A) A real-time graph of oxygen consumption rate (OCR) at basal respiration followed by sequential injection of SHAM (2 mM), Oligomycin (10 &#181;M), KCN (10 mM), and Antimycin A (2 &#181;M). B) Comparison of area under the curve (AUC) basal OCR between wild type, mam33&#916;/&#916;, and mam33&#916;/&#916;::MAM33 strains of C. albicans show no significant difference. C) A real-time graph of extracellular acidification rate (ECAR) at basal glycolysis followed by sequential injection of SHAM (2 mM), Oligomycin (10 &#181;M), KCN (10 mM), and Antimycin A (2 &#181;M). D) Comparison of area under the curve (AUC) basal ECAR between wild type, mam33&#916;/&#916;, and mam33&#916;/&#916;::MAM33 strains of C. albicans show no significant difference. E) Comparison of area under curve (AUC) ECAR between wild type, mam33&#916;/&#916; and mam33 &#916;/&#916;::MAM33 strains of C. albicans after the injection of C-IV inhibitor KCN show significant decrease in glycolytic shift in mam33&#916;/&#916; mutant compared to wild type and complementary strains Values are expressed as average &#177; standard error mean. * p&#60;0.05, ** p&#60;0.001 is considered as significant by one-way ANOVA. <a href="https://www.jove.com/files/ftp_upload/58913/58913fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58913/58913fig8.jpg" /> 
Figure 8. Glycolytic stress assay in C. albicans. Oxygen consumption and extracellular acidification were measured in wild type, mam33&#916;/&#916; mutant after they were starved for glucose followed by acute injection of saturated glucose concentration. A) A real-time graph of oxygen consumption rate after cells were subjected to basal ECAR followed by sequential injection of glucose (10 mM), Oligomycin (1 &#181;M), 2-DG (40 mM), and Antimycin A (2 &#181;M). B) Bar graph showing the area under the curve ECAR of basal glycolysis between wild type, mam33&#916;/&#916;, and mam33&#916;/&#916;::MAM33 strains of C. albicans. C) A real-time graph of the glycolytic function after cells were subjected to basal ECAR followed by sequential injection of glucose (10 mM), Oligomycin (1 &#181;M), 2-DG (40mM), and Antimycin A (2 &#181;M). D) Bar graph showing the area under the curve ECAR of basal glycolysis between wild type, mam33&#916;/&#916;, and mam33&#916;/&#916;::MAM33 strains of C. albicans Values are expressed as average &#177; standard error mean. * p&#60;0.05, *** p&#60;0.0005, **** p&#60;0.0001 is considered as significant by one-way ANOVA. <a href="https://www.jove.com/files/ftp_upload/58913/58913fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis at JoVE.com

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

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

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

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular ...

bio-energetics-investigation-candida-albicans-using-real-time

Nanyang Technological University

Research

JoVE Journal

Medicine

6.9K Views.  The State University of New Jersey. Here, we present a stepwise protocol to investigate the mitochondrial respiration and glycolytic function in Candida Albicans using an extra flux analyzer.

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

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third most common infection in US hospitals. Different manifestations of candidiasis are associated with biofilm formation, both on host tissues and/or medical devices (i.e. catheters). Biofilm formation carries negative clinical implications, as cells within the biofilms are protected from host immune responses and from the action of antifungals. We have developed a simple, fast and robust in vitro model for the formation of C. albicans biofilms using 96 well microtiter-plates, which can also be used for biofilm antifungal susceptibility testing. The readout of this assay is colorimetric, based on the reduction of XTT (a tetrazolium salt) by metabolically active fungal biofilm cells. A typical experiment takes approximately 24 h for biofilm formation, with an additional 24 h for antifungal susceptibility testing. Because of its simplicity and the use of commonly available laboratory materials and equipment, this technique democratizes biofilm research and represents an important step towards the standardization of antifungal susceptibility testing of fungal biofilms.

A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

We describe a simple, rapid and robust method for the formation of Candida albicans biofilms using 96 well microtiter plates and its utility in antifungal susceptibility testing of cells within biofilms.

Candida albicans remains the most frequent cause of fungal infections in an expanding population of compromised patients and candidiasis is now the third ...

Immunology and Infection

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Candida albicans remains the main etiological agent of candidiasis, which currently represents the fourth most common nosocomial bloodstream infection in US hospitals1. These opportunistic infections pose a growing threat for an increasing number of compromised individuals, and carry unacceptably high mortality rates. This is in part due to the limited arsenal of antifungal drugs, but also to the emergence of resistance against the most commonly used antifungal agents. Further complicating treatment is the fact that a majority of manifestations of candidiasis are associated with the formation of biofilms, and cells within these biofilms show increased levels of resistance to most clinically-used antifungal agents2. Here we describe the development of a high-density microarray that consists of C. albicans nano-biofilms, which we have named CaBChip3. Briefly, a robotic microarrayer is used to print yeast cells of C. albicans onto a solid substrate. During printing, the yeast cells are enclosed in a three dimensional matrix using a volume as low as 50 nL and immobilized on a glass substrate with a suitable coating. After initial printing, the slides are incubated at 37 &deg;C for 24 hours to allow for biofilm development. During this period the spots grow into fully developed "nano-biofilms" that display typical structural and phenotypic characteristics associated with mature C. albicans biofilms (i.e. morphological complexity, three dimensional architecture and drug resistance)4. Overall, the CaBChip is composed of ~750 equivalent and spatially distinct biofilms; with the additional advantage that multiple chips can be printed and processed simultaneously. Cell viability is estimated by measuring the fluorescent intensity of FUN1 metabolic stain using a microarray scanner. This fungal chip is ideally suited for use in true high-throughput screening for antifungal drug discovery. Compared to current standards (i.e. the 96-well microtiter plate model of biofilm formation5), the main advantages of the fungal biofilm chip are automation, miniaturization, savings in amount and cost of reagents and analyses time, as well as the elimination of labor intensive steps. We believe that such chip will significantly speed up the antifungal drug discovery process.

Candida albicans Biofilm Chip CaBChip for High-throughput Antifungal Drug Screening

We have developed a high-density microarray platform consisting of 3D nano-biofilms of C. albicans called CaBChip. The susceptibility profile of drugs tested on a CaBChip is comparable to the conventional 96-well plate model, suggesting that the fungal chip is ideally suited for true high-throughput screening of antifungal drugs.

Candida  albicans remains the main etiological agent of candidiasis, which  currently represents the fourth most common nosocomial bloodstream infection ...

Bioengineering

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. While models for the growth under flow have been developed, many of these systems are expensive, or do not allow imaging while the cells are under flow. We have developed a novel apparatus that allows us to image the growth and development of Candida albicans cells under flow and in real-time. Here, we detail the protocol for the assembly and use of this flow apparatus, as well as the quantification of data that are generated. We are able to quantify the rates that the cells attach to and detach from the slide, as well as to determine a measure of the biomass on the slide over time. This system is both economical and versatile, working with many types of light microscopes, including inexpensive benchtop microscopes, and is capable of extended imaging times compared to other flow systems. Overall, this is a low-throughput system that can provide highly detailed real-time information on the biofilm growth of fungal species under flow.

We describe the assembly, operation, and cleaning of a flow apparatus designed to image fungal biofilm formation in real time while under flow. We also provide and discuss quantitative algorithms to be used on the acquired images.

In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut.

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

Candida albicans is an important human pathogen. Its ability to switch between morphologic forms is central to&#160;its pathogenesis; these morphologic changes are regulated by a complex signaling network controlled in response to environmental stimuli. These regulatory components have been highly studied, but almost all studies use a variety of in vitro stimuli to trigger filamentation. To determine how morphogenesis is regulated during the pathogenesis process, we developed an in vivo microscopy system to obtain high spatial resolution images of organisms undergoing hyphal formation within the mammalian host. The protocol presented here describes the use of this system to screen small collections of C. albicans mutant strains, allowing us to identify key regulators of morphogenesis as it occurs at the site of infection. Representative results are presented, demonstrating that some regulators of morphogenesis, such as the transcriptional regulator Efg1, have consistent phenotypes in vitro and in vivo, whereas other regulators, such as adenyl cyclase (Cyr1), have significantly different phenotypes in vivo compared to in vitro.

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

This manuscript describes a method for screening moderately sized Candida albicans mutant libraries for morphogenesis phenotypes during active infection in a mammalian host using non-invasive confocal microscopy.

Candida albicans is an important human pathogen. Its ability to switch between morphologic forms is central to&#160;its pathogenesis; these morphologic ...

Ethanol Impairs AM Bioenergetics by Shifting to Glutamine-Dependent Respiration

Assessment of Glutamine as a Fuel Source for Alveolar Macrophages Exposed to Chronic Ethanol Using an Extracellular Flux Bioanalyzer

Alveolar macrophages (AMs) are the first line of cellular defense in the lower airway against pathogens. However, chronic and excessive alcohol use impairs the ability of AMs to phagocytize and clear pathogens from the alveolar space, in part through dysregulated fuel metabolism and bioenergetics. Our prior work has shown that chronic ethanol (EtOH) consumption impairs mitochondrial bioenergetics and increases lactate levels in AMs. Further, we recently demonstrated that EtOH increases glutamine dependency and glutamine-dependent maximal respiration while decreasing flexibility, shifting away from pyruvate-dependent respiration and towards glutamine-dependent respiration. Glutaminolysis is an important compensatory pathway for mitochondrial respiration when pyruvate is used for lactic acid production or when other fuel sources are insufficient. Using a mouse AM cell line, MH-S cells, exposed to either no EtOH or EtOH (0.08%) for 72 h, we determined the dependency of mitochondrial respiration and bioenergetics on glutamine as a fuel source using an extracellular flux bioanalyzer. Real-time measures were done in response to bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl) ethyl sulfide (BPTES), an inhibitor of glutaminase 1, which prevents the enzymatic conversion of glutamine to glutamate, in media vehicle or in response to vehicle alone, followed by testing mitochondrial stress. The step-by-step protocol provided herein describes our methods and calculations for analyzing average levels of glutamine-dependent basal mitochondrial respiration, mitochondrial ATP-linked respiration, maximal mitochondrial respiration, and mitochondrial spare respiratory capacity across multiple biological and experimental replicates.

Alcohol misuse impairs alveolar macrophage (AM) immunity due to suppressed mitochondrial respiration and bioenergetics. We recently demonstrated that ethanol (EtOH) exposure increases glutamine dependency for mitochondrial respiration in AMs. Herein, methods are provided to determine the usage of glutamine for mitochondrial respiration in EtOH-treated AMs using an extracellular flux bioanalyzer.

Alveolar macrophages (AMs) are the first line of cellular defense in the lower airway against pathogens. However, chronic and excessive alcohol use ...

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over the past two decades, we have developed a yeast membrane protein expression platform that has been used to study many important fungal membrane proteins. The expression host Saccharomyces cerevisiae AD&#916;&#916; is deleted in seven major endogenous ABC transporters and it contains the transcription factor Pdr1-3 with a gain-of-function mutation that enables the constitutive overexpression of heterologous membrane protein genes stably integrated as single copies at the genomic PDR5 locus. The creation of versatile plasmid vectors and the optimization of one-step cloning strategies enables the rapid and accurate cloning, mutagenesis, and expression of heterologous ABC transporters. Here, we describe the development and use of a novel protease-cleavable mGFPHis double tag (i.e., the monomeric yeast enhanced green fluorescent protein yEGFP3 fused to a six-histidine affinity purification tag) that was designed to avoid possible interference of the tag with the protein of interest and to increase the binding efficiency of the His tag to nickel-affinity resins. The fusion of mGFPHis to the membrane protein ORF (open reading frame) enables easy quantification of the protein by inspection of polyacrylamide gels and detection of degradation products retaining the mGFPHis tag. We demonstrate how this feature facilitates detergent screening for membrane protein solubilization. A protocol for the efficient, fast, and reliable isolation of the small-scale plasma membrane preparations of the C-terminally tagged Candida albicans multidrug efflux transporter Cdr1 overexpressed in S. cerevisiae AD&#916;&#916;, is presented. This small-scale plasma membrane isolation protocol generates high-quality plasma membranes within a single working day. The plasma membrane preparations can be used to determine the enzyme activities of Cdr1 and Cdr1 mutant variants.

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

This article presents a small-scale plasma membrane isolation protocol for the characterization of Candida albicans ABC (ATP-binding cassette) protein Cdr1, overexpressed in Saccharomyces cerevisiae. A protease-cleavable C-terminal mGFPHis double tag with a 16-residue linker between Cdr1 and the tag was designed to facilitate the purification and detergent-screening of Cdr1.

The successful biochemical and biophysical characterization of ABC transporters depends heavily on the choice of the heterologous expression system. Over ...

Biology

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, prevent fracture. In the interest to both optimize peak bone mass and combat various musculoskeletal diseases (i.e., post-menopausal osteoporosis, anorexia nervosa, type 1 and 2 diabetes mellitus), incredible efforts have been made in the field of bone biology to fully characterize osteoblasts throughout their differentiation process. Given the primary role of mature osteoblasts to secrete matrix proteins and mineralization vesicles, it has been noted that these processes take an incredible amount of cellular energy, or adenosine triphosphate (ATP). The overall cellular energy status is often referred to as cellular bioenergetics, and it includes a series of metabolic reactions that sense substrate availability to derive ATP to meet cellular needs. Therefore, the current method details the process of isolating primary, murine bone marrow stromal cells (BMSCs) and monitoring their bioenergetic status using the Real-time cell metabolic flux analyzer at various stages in osteoblast differentiation. Importantly, these data have demonstrated that the metabolic profile changes dramatically throughout osteoblast differentiation. Thus, using this physiologically relevant cell type is required to fully appreciate how a cell's bioenergetic status can regulate the overall function.

Real-time cell metabolic flux assay measures the oxygen consumption rate and extracellular acidification rate, which corresponds to mitochondrial and glycolytic adenosine triphosphate production, using pH and oxygen sensors. The manuscript explains a method to understand the energy status of osteoblasts and the characterization and interpretation of the cellular bioenergetic status.

Bone formation by osteoblasts is an essential process for proper bone acquisition and bone turnover to maintain skeletal homeostasis, and ultimately, ...

Extracellular Flux Assay: A Method for Measuring Metabolic Profile of Cells

Source: Hlozkova K. et al. <a href="https://www.jove.com/t/58426">Assessment of the Metabolic Profile of Primary Leukemia Cells.</a> J. Vis. Exp. (2018).
This protocol provides a biochemical approach for measuring extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of primary leukemic cells with extracellular flux assay. The assessment of the metabolic profile of these cells helps to characterize their demands, leading to personalized therapy.

Test strumienia zewnątrzkomórkowego: metoda pomiaru profilu metabolicznego komórek

Source: Hlozkova K. et al. Assessment of the Metabolic Profile of Primary Leukemia Cells. J. Vis. Exp. (2018).
This protocol provides a biochemical ...

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

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A 96 Well Microtiter Plate-based Method for Monitoring Formation and Antifungal Susceptibility Testing of Candida albicans Biofilms

Candida albicans Biofilm Chip (CaBChip) for High-throughput Antifungal Drug Screening

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

Assessment of Glutamine as a Fuel Source for Alveolar Macrophages Exposed to Chronic Ethanol Using an Extracellular Flux Bioanalyzer

Small-Scale Plasma Membrane Preparation for the Analysis of Candida albicans Cdr1-mGFPHis

Using Real-Time Cell Metabolic Flux Analyzer to Monitor Osteoblast Bioenergetics

Test strumienia zewnątrzkomórkowego: metoda pomiaru profilu metabolicznego komórek