Aequorin as a Useful Calcium-Sensing Reporter in Candida albicans

In Candida albicans, calcium ions (Ca²⁺) regulate the activity of several signaling pathways, especially the calcineurin signaling pathway. Ca²⁺ 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 Ca²⁺ homeostasis can be addressed in this fungal pathogen. Aequorin from Aequorea victoria has been used in eukaryotic cells for detecting intracellular Ca²⁺. A codon-adapted aequorin Ca²⁺-sensing expression system was therefore designed for probing cytosolic Ca²⁺ 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 Ca²⁺ levels in C. albicans. Alkaline stress resulted in an immediate influx of Ca²⁺ from the extracellular medium. This increase was exacerbated in a mutant lacking the vacuolar Ca²⁺ transporter VCX1, thus confirming its role in Ca²⁺ homeostasis. Using mutants in components of a principal Ca²⁺ channel (MID1, CCH1), the alkaline-dependent Ca²⁺ spike was greatly reduced, thus highlighting the crucial role of this channel complex in Ca²⁺ uptake and homeostasis. Exposure to the antiarrhythmic drug amiodarone, known to perturb Ca²⁺ trafficking, resulted in increased cytoplasmic Ca²⁺ within seconds that was abrogated by the chelation of Ca²⁺ in the external medium. Ca²⁺ import was also dependent on the Cch1/Mid1 Ca²⁺ channel in amiodarone-exposed cells. In conclusion, the aequorin Ca²⁺ sensing reporter developed here is an adequate tool with which Ca²⁺ homeostasis can be investigated in C. albicans.