Dynamic calcium-mediated stress response and recovery signatures in the fungal pathogen, Candida albicans .

Calcium (Ca 2+ ) is an important second messenger for activating stress response signaling and cell adaptation in eukaryotic cells yet intracellular Ca 2+ -dynamics in fungi are poorly understood due to lack of effective real-time Ca 2+ reporters. We engineered the GCaMP6f construct for use in the fungal pathogen, Candida albicans , and used live-cell imaging to observe both dynamic Ca 2+ spiking and slower changes in non-spiking Ca 2+ -GCaMP signals elicited by stress or gene deletion. Short-term exposure to membrane, osmotic or oxidative stress generated immediate stress-specific responses and repeated exposure revealed differential recovery signatures. Osmotic stress caused yeast cell shrinkage and no adaptation response, where Ca 2+ -GCaMP spiking was inhibited by 1 M NaCl but not by 0.666 M CaCl 2. Treatment with sodium dodecylsulfate (SDS) caused a spike-burst, raised the non-spiking Ca 2+ -GCaMP signals, and caused significant cell death, but surviving cells adapted over subsequent exposures. Treatment with 5 mM H 2 O 2 abolished spiking and caused transient non-GCaMP-related autofluorescence, but cells adapted such that spiking returned and autofluorescence diminished on repeated exposure. Adaptation to H 2 O 2 was dependent on Cap1, extracellular Ca 2+ , and calcineurin but not on its downstream target, Crz1. Ca 2+ -dynamics were not affected by H 2 O 2 in the hog1 Δ or yvc1 Δ mutants, suggesting a pre-adapted, resistant state, possibly due to changes in membrane permeability. Live-cell imaging of Ca 2+ -GCaMP responses in individual cells has, therefore, revealed the dynamics of Ca 2+ -influx, signaling and homeostasis, and their role in the temporal stress response signatures of C. albicans . IMPORTANCE Intracellular calcium signaling plays an important role in the resistance and adaptation to stresses encountered by fungal pathogens within the host. This study reports the optimization of the GCaMP fluorescent calcium reporter for live-cell imaging of dynamic calcium responses in single cells of the pathogen, Candida albicans , for the first time. Exposure to membrane, osmotic or oxidative stress generated both specific changes in single cell intracellular calcium spiking and longer calcium transients across the population. Repeated treatments showed that calcium dynamics become unaffected by some stresses but not others, consistent with known cell adaptation mechanisms. By expressing GCaMP in mutant strains and tracking the viability of individual cells over time, the relative contributions of key signaling pathways to calcium flux, stress adaptation, and cell death were demonstrated. This reporter, therefore, permits the study of calcium dynamics, homeostasis, and signaling in C. albicans at a previously unattainable level of detail.