Functional Study Of Calcium Homeostasis System In Oxidative Stress Response In Candida Albicans

Candida albicans, the major human fungal pathogen, causes a range of disorders from mild infections to life-threatening disease. Nowadays, with the widespread use of immunosuppressive therapy, infections by C. albicans have become more common and more studies about this pathogen are needed.Like other eukaryotic cells, C. albicans cells respond and adapt to environmental stresses by evolutionarily conserved multicomponent endogenous systems that utilize a network of signal transduction pathways to regulate the adaptive and protective phenotype. Ca2+-mediated signaling of stress conditions is used by virtually every eukaryotic cell to regulate a wide variety of cellular processes through transient increases in cytosolic Ca2+. Fluctuations in intracellular Ca2+levels are known to initiate responses to environmental stimuli in a wide variety of cell types. One of the principal mediators of this Ca2+signal in eukaryotic cells is calmodulin, a small Ca2+-binding protein. Upon binding Ca2+, calmodulin then changes its conformation, forming the Ca2+-calmodulin complex that controls the activity of several key regulatory enzymes. In Saccharomyces cerevisiae, notable Ca+-calmodulin targets are calcineurin and the type Ⅱ Ca2+-calmodulin dependent protein kinases (CaMKs). CaMKs are responsible for decoding intracellular Ca2+ion fluctuation in terms of a Ca2+-mediated physiological response. Although many members of the calcium homeostasis system in C. albicans have been elucidated, more studies are still needed, since cytosolic free Ca2+levels rise and fall rapidly with complex dynamics, which initiate cellular regulation in response to environmental stimuli. The main results in the study were demonstrated as follows.(1) By BLASTP analysis, we identified the homologue of S. cerevisiae ECM7in the Candida Genome Database. We constructed an ecm7null mutant strain and found that Ecm7as a putative regulator of high affinity Ca2+influx system (HACS) plays an important role in calcium homeostasis maintenance and is stabilized mainly by Midi. Disruption of ECM7led to increased sensitivity to calcium-depleted conditions. Flow cytometry analysis revealed that Ecm7mediated Ca2+influx under high pH shock. Cycloheximide chase experiments further showed that MIDI deletion significantly decreased the stability of Ecm7. Besides, ecm7△/△cells were hypersensitive to oxidative stress. ECM7deletion not only led to the down-regulation of oxidative stress response gene, but also induced the degradation of Cap1when exposed to H2O2treatment. The ecm7△/△mutant also showed a defect in hyphal development, which was accompanied with the decreased expression of hyphal related gene HWP1. Though subsequent experiments revealed that the ecm7△/△mutant showed similar virulence to the wild-type strain, the ability of invasion and diffusion of the mutant in mouse kidneys decreased.(2) To further investigate the roles of the calcium homeostasis system, we also constructed the CaMK mutants. They are the cmkl△/△, the cmk2△/△and the cmk1△/△cmk2△/△mutants. We found that CaMKs function in cell wall integrity (CWI) and cellular redox regulation. Loss of either CMK1or CMK2, or both resulted in increased expression of CWI-related genes under Calcofluor white (CFW) treatment. Besides, CaMKs are essential for the maintenance of cellular redox balance. Disruption of either CMK1or CMK2, or both not only led to a significant increase of intracellular ROS levels, but also led to a decrease of the mitochondrial membrane potential (MMP), suggesting the important roles that CaMKs play in the maintenance of the mitochondrial function.