白色念珠菌高分辨率呼吸测定

Lucian  Duvenage; Carol A. Munro; Campbell  W. Gourlay

doi:10.21769/BioProtoc.3361

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Peer-reviewed

High Resolution Respirometry in Candida albicans

白色念珠菌高分辨率呼吸测定

LD Lucian Duvenage

CM Carol A. Munro

Campbell W. Gourlay email

发布: 2019年09月05日第9卷第17期 DOI: 10.21769/BioProtoc.3361 浏览次数: 3867

评审: Emily CopeEmmanuel Orta-ZavalzaLucy Xie

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Abstract

Many Candida species, such as the opportunistic human pathogen Candida albicans, are Crabtree-Negative yeasts and are therefore highly dependent on the energy generated through oxidative phosphorylation. Respiration control is linked to a range of aspects of C. albicans cell physiology that appear to be important for virulence, most notably its ability to switch from yeast to hyphal forms and the maintenance of the cell wall. The following protocol allows for the measurement and characterization of respiration in C. albicans using high resolution respirometry. We outline how addition of respiration inhibitors can be used to assay the “mode” of respiration, mitochondrial health and the level of electron transport that is coupled to ATP synthase activity in living cell cultures. These data provide useful insight into the effects of external factors, such as exposure to anti-fungal compounds, or internal changes such as genetic alterations on respiratory performance.

Keywords: Candida (念珠菌)

Background

In the human fungal pathogen Candida albicans, respiration plays a key role in the yeast-to-hypha transition (Grahl et al., 2015), the catabolism of morphogenic amino acids and escape from macrophages (Silao et al., 2019). Respiration inhibitors may be considered for use as antifungals, as they can limit growth, promote cell death and in some cases act in co-operation with current antifungals such as fluconazole (Vincent et al., 2016). Therefore, characterization of C. albicans respiration in response to different environments, nutrients and inhibitors is important to understand pathogenicity and may also prove useful for future drug development.

C. albicans possesses a classical electron transport chain consisting of Complexes I-IV, as well as cyanide-insensitive alternative oxidases (Helmerhorst et al., 2002) and a less well defined parallel pathway (Duvenage et al., 2019). The level of AOX activity increases upon inhibition of classical electron chain components, allowing respiration to continue (Huh and Kang, 2001). C. albicans are still able to respire, albeit at a much reduced level, upon additional inhibition of AOX via the parallel pathway. C. albicans therefore contain a highly robust respiratory network that can resist inhibition.

Analysis of respiration in whole yeast cells can yield physiologically relevant information, such as integration with cellular processes that is not captured when analyzing electron transport within isolated mitochondria. Oxygen consumption in yeast is commonly measured using a Clarke oxygen electrode or chemical extracellular oxygen assays. However, a graphical readout of oxygen flux in real time using high-resolution respirometry can give important information on time-critical events, such as the induction of alternative respiration, and allows for adaptation of the experiment to include more complex titration schedules (Hütter et al., 2006). The following protocol describes the measurement of respiration in yeast-form cells of C. albicans (and other Candida species) in real time, for the characterization of mitochondrial function and the responses to added inhibitors.

Materials and Reagents

Culture tubes (Falcon 50 ml conical centrifuge cubes, Fisher Scientific, catalog number: 14-432-22)
Microcentrifuge tubes, 1.5 ml (Sigma-Aldrich, catalog number: Z606340)
Cuvettes (Fisher Scientific, catalog number: 11547692)
Microlitre syringes (Hamilton^®: 10 µl- and 25 µl microlitre syringes, Sigma-Aldrich, catalog numbers: 28615U, 20735)
Candida albicans (e.g., SC5314) overnight culture in YPD
Triethyltin bromide (TET) (Sigma-Aldrich, catalog number: 288047)
FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone) (Sigma-Aldrich, catalog number: C2920)
Potassium cyanide (KCN) (Sigma-Aldrich, catalog number: 60178)
Salicylhydroxamic acid (SHAM) (Sigma-Aldrich, catalog number: S607)
Bacto peptone (Fisher Scientific, catalog number: S71604)
Yeast extract (Fisher Scientific, catalog number: B11929)
Glucose (Sigma-Aldrich, catalog number: G8270)
NaCl (Sigma-Aldrich, catalog number: AlS7653)
KCl (Sigma-Aldrich, catalog number: P9333)
Na₂HPO₄(Sigma-Aldrich, catalog number: S7907)
KH₂PO₄ (Sigma-Aldrich, catalog number: P9791)
HCl (Sigma-Aldrich, catalog number: H1758)
Antimycin A
Sodium nitroprusside
Yeast extract-peptone media, 2% glucose (YPD) (see Recipes)
Phosphate buffered saline (PBS) (see Recipes)
Note: 50 mM TET, 2 mM FCCP, 2 mM Antimycin A and 200 mM SHAM stocks are prepared in absolute ethanol. 1 M KCN stocks are prepared in water. Aliquots should be prepared and stored at -20 °C. Respiration inhibitors are toxic and appropriate safety measures should be taken during preparation, handling and storage.

Equipment

Oxygraph-2k respirometer (O2k-core) (Oroboros Instruments Corp, Austria)
Incubator (e.g., New Brunswick^TM Innova^® 44, Eppendorf, catalog number: M1282-0002)
Spectrophotometer (e.g., Jenway^TM 6305 UV/Visible Spectrophotometer, Fisher Scientific, catalog number: 11400539)
Microcentrifuge (e.g., centrifuge 5418, Eppendorf, catalog number: 5418000017)
Haemocytometer (Sigma-Aldrich, BR718605)
Optical microscope (e.g., Cole-Parmer, catalog number: UY-48922-70)
Autoclave (e.g., Astell Scientific^TM benchtop autoclave, Fisher Scientific, catalog number: 12735375)

Software

Datlab 6 (or more recent release) software (Oroboros Instruments Corp)

Procedure

English

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Duvenage, L., Munro, C. A. and Gourlay, C. W. (2019). High Resolution Respirometry in Candida albicans. Bio-protocol 9(17): e3361. DOI: 10.21769/BioProtoc.3361.