Aequorin as a Useful Calcium-Sensing Reporter in Candida albicans

In Candida albicans, calcium ions (Ca(2+)) regulate the activity of several signaling pathways, especially the calcineurin signaling pathway. Ca(2+) homeostasis is also important for cell polarization, hyphal extension, and plays a role in contact sensing. It is therefore important to obtain accurate tools with which Ca(2+) homeostasis can be addressed in this fungal pathogen. Aequorin from Aequorea victoria has been used in eukaryotic cells for detecting intracellular Ca(2+). A codon-adapted aequorin Ca(2+)-sensing expression system was therefore designed for probing cytosolic Ca(2+) flux in C. albicans. The availability of a novel water-soluble formulation of coelenterazine, which is required as a co-factor, made it possible to measure bioluminescence as a readout of intracellular Ca(2+) levels in C. albicans. Alkaline stress resulted in an immediate influx of Ca(2+) from the extracellular medium. This increase was exacerbated in a mutant lacking the vacuolar Ca(2+) transporter VCX1, thus confirming its role in Ca(2+) homeostasis. Using mutants in components of a principal Ca(2+) channel (MID1, CCH1), the alkaline-dependent Ca(2+) spike was greatly reduced, thus highlighting the crucial role of this channel complex in Ca(2+) uptake and homeostasis. Exposure to the antiarrhythmic drug amiodarone, known to perturb Ca(2+) trafficking, resulted in increased cytoplasmic Ca(2+) within seconds that was abrogated by the chelation of Ca(2+) in the external medium. Ca(2+) import was also dependent on the Cch1/Mid1 Ca(2+) channel in amiodarone-exposed cells. In conclusion, the aequorin Ca(2+) sensing reporter developed here is an adequate tool with which Ca(2+) homeostasis can be investigated in C. albicans.