Calcineurin-mediated intracellular organelle calcium homeostasis is required for the survival of fungal pathogens upon extracellular calcium stimuli

Abstract

In eukaryotes, calcium not only is an essential mineral nutrient but also serves as an intracellular second messenger that is necessary for many physiological processes. Previous studies showed that the protein phosphatase-calcineurin protects fungi from toxicity caused by the extracellular calcium; however, little is known about how calcineurin mediates the cellular physiology process for this function. In this study, by monitoring intracellular calcium, particularly by tracking vacuolar calcium dynamics in living cells through a novel procedure using modified aequorin, we found that calcineurin dysfunction systematically caused abnormal intracellular calcium homeostasis in cytosol, mitochondria, and vacuole, leading to drastic autophagy, global organelle fragmentation accompanied with the increased expression of cell death-related enzymes, and cell death upon extracellular calcium stimuli. Notably, all detectable defective phenotypes seen with calcineurin mutants can be significantly suppressed by alleviating a cytosolic calcium overload or increasing vacuolar calcium storage capacity, suggesting toxicity of exogenous calcium to calcineurin mutants is tightly associated with abnormal cytosolic calcium accumulation and vacuolar calcium storage capacity deficiency. Our findings provide insights into how the original recognized antifungal drug target-calcineurin regulates intracellular calcium homeostasis for cell survival and may have important implications for antifungal therapy and clinical drug administration.

Keywords: Fungi; aspergillus fumigatus; autophagy; calcineurin; calcium homeostasis; pathogenic fungi.

Figure 1.

Calcium is toxic to calcineurin mutants

Figure 2.

Deletion of cnaA affects intracellular calcium accumulation in response to extracellular calcium stimuli

Figure 3.

The loss of CnaA causes vacuolar fragmentation and atg2-related autophagy in the presence of excess calcium

Figure 4.

Deletion of cchA alleviates the calcium toxicity-related phenotypes in the ΔcnaA mutant

Figure 5.

Deletion of cchA in the ΔcnaA mutant did not restore the virulence in a mouse infection model

Figure 6.

Overexpressing P-type Ca²⁺-ATPase PmcA in the ΔcnaA mutant significantly rescues calcium toxicity-related phenotypes

Figure 7.

ΔcnaA mutant shows global fragmentation of nuclei and organelles upon calcium stimuli