The role of calcium signaling pathway and vacuoles in cell death

Fungal resistance to currently available antibiotics is a growing problem worldwide. Recent studies have shown that most antifungal drugs used clinically are only fungistatic, not fungicidal, because they activate a calcium-dependent anti-death pathway. The main component of this pathway is a phosphatase calcineurin (Cn), and inhibition of Cn by immunosuppressants FK506 or Cyclosporin A (CsA) blocks the anti-death pathway and makes antifungal drugs lethal. Another component of this pathway is Ca2+/calmodulin-dependent kinase II (Cmk2), which has an anti-death role independent of Cn. We developed and performed a genetic screen to identify downstream effectors of CN that mimic this anti-death function in response to a natural antifungal tunicamycin. Although the screen did not yield a direct substrate of Cn, it revealed components and events that are important in cell death. We found that an endocytic protein Ede1 functions downstream of Cmk2 and possesses the same role in death and Ca2+ uptake. Protein kinase A (PKA) in the cAMP pathway was identified as a potential pro-death kinase that may counteract the anti-death phosphatase Cn. We also show that the vacuoles are important in death in multiple aspects: (1) the two main pro-death factors required for death were related to vacuoles (i.e. the vacuolar H+-ATPase, which acidifies the organelles, and TORC1 in the target of rapamycin (TOR) pathway, which gets recruited to vacuoles when activated by nutrients), (2) the vacuoles show distinct morphology during death in response to stimuli, and (3) the vacuolar membrane permeabilizes during death and both the morphological change and the permeabilization are dependent on the V-ATPase.;We also show that the anti-death role of the Ca2+ signaling pathway and the pro-death role of the V-ATPase are conserved in the infectious fungi Candida albicans and Candida glabrata in response to clinically used azole-class antifungal drugs.