The Cch1-Mid1 High-Affinity Calcium Channel Contributes to the Virulence of Cryptococcus neoformans by Mitigating Oxidative Stress

Abstract

bPathogenic fungi have developed mechanisms to cope with stresses imposed by hosts. For Cryptococcus spp., this implies active defense mechanisms that attenuate and ultimately overcome the onslaught of oxidative stresses in macrophages. Among cellular pathways within Cryptococcus neoformans' arsenal is the plasma membrane high-affinity Cch1-Mid1 calcium (Ca 2؉ ) channel (CMC). Here we show that CMC has an unexpectedly complex and disparate role in mitigating oxidative stress. Upon inhibiting the Ccp1-mediated oxidative response pathway with antimycin, strains of C. neoformans expressing only Mid1 displayed enhanced growth, but this was significantly attenuated upon H 2 O 2 exposure in the absence of Mid1, suggesting a regulatory role for Mid1 acting through the Ccp1-mediated oxidative stress response. This notion is further supported by the interaction detected between Mid1 and Ccp1 (cytochrome c peroxidase). In contrast, Cch1 appears to have a more general role in promoting cryptococci survival during oxidative stress. A strain lacking Cch1 displayed a growth defect in the presence of H 2 O 2 without BAPTA [(1,2-bis(2-aminophenoxy)ethane-N,N,N=,N=-tetraacetic acid, cesium salt] or additional stressors such as antimycin. Consistent with a greater contribution of Cch1 to oxidative stress tolerance, an intracellular growth defect was observed for the cch1⌬ strain in the macrophage cell line J774A.1. Interestingly, while the absence of either Mid1 or Cch1 significantly compromises the ability of C. neoformans to tolerate oxidative stress, the absence of both Mid1 and Cch1 has a negligible effect on C. neoformans growth during H 2 O 2 stress, suggesting the existence of a compensatory mechanism that becomes active in the absence of CMC. I t is well established that Ca2ϩ is a critical secondary messenger that initiates and regulates a plethora of signaling events. For this reason, cytosolic Ca 2ϩ levels are exquisitely controlled by regulating the movement of calcium ions into and out of cells via ion channels and transporters (1-3). Fluctuations of Ca 2ϩ in the cytosol are transduced via calcium sensors like calmodulin, which, upon calcium binding, activates calcineurin and CaMK (Ca 2ϩ / calmodulin-dependent protein kinases). Calcineurin is a Ca 2ϩ / calmodulin-activated serine/threonine protein phosphatase highly conserved among eukaryotes. In fungi such as Cryptococcus neoformans, Candida albicans, and Saccharomyces cerevisiae, calcineurin regulates the transcription of genes involved in mating, cell viability, and response to cell stress (4-6). The improper regulation of Ca 2ϩ can produce significant cell damage and ultimately lead to cell death (7).In fungal cells, the Cch1-Mid1 channel complex (CMC) represents the only high-affinity Ca 2ϩ channel in the plasma membrane that mediates the specific influx of Ca 2ϩ (2). While Cch1 functions as the pore of the channel, Mid1 associates with Cch1 and, in a manner that is not completely understood, facilitates the movement of Ca 2ϩ from the extracellular milieu to the ...

Calcium channel regulator Mid1 links TORC2-mediated changes in mitochondrial respiration to autophagy

“…We next examined a role for the conserved high-affinity calcium uptake system (HACS) responsible for importing extracellular calcium into the cell (Ton and Rao, 2004; Martin et al, 2011). The HACS regulates calcium import through a plasma membrane (PM)-localized calcium channel formed by the proteins Cch1 and Mid1 (Iida et al, 1994; Ozeki-Miyawaki et al, 2005; Martin et al, 2011; Vu et al, 2015). Interestingly, we observed that deletion of Mid1, but not Cch1, was sufficient to return calcineurin activity to basal WT levels in ypk1Δ cells (Fig.…”

“…The C-terminal domain of Mid1 contains several phylogenetically conserved cysteine residues that have the potential to be oxidized during conditions of oxidative stress (Finkel, 2011; Funato and Miki, 2013; Vu et al, 2015). To begin to explore the mechanism by which Mid1 is regulated by mitochondrial ROS, we asked whether this cysteine-rich domain is important for Mid1 activity in Ypk1-deficient cells.…”

Calcium binding protein Ncs1 is calcineurin-regulated inCryptococcus neoformansand essential for cell division and virulence

“…However, expression of both genes increased by approximately 3-fold in the ncs1∆ mutant strain following growth in the presence of Ca 2+ (Fig 3B). This suggests that Cch1 and Mid1 could be the potential source of the extra Ca 2+ in the ncs1∆ mutant, since they import Ca 2+ to the cytosol (29,47). We also generated a mid1∆ncs1∆ double mutant in C. neoformans to evaluate if calcium sensitivity would be restored.…”

Cryptococcus and Phagocytes: Complex Interactions that Influence Disease Outcome

“…Cryptococcus neoformans is capable of surviving even in a phagosome with ROS, in part due to absorption of ROS by the capsule ( Zaragoza et al, 2008 ). In addition, the plasma membrane high-affinity Cch1-Mid1 calcium channel (CMC) of C. neoformans promotes cryptococcal survival during exposure to oxidative stress ( Vu et al, 2015 ). Cch1 mutants have decreased survival in J774.1A macrophages, however, C. neoformans deficient in both Cch1 and Mid1 maintain resistance to the ROS, suggesting a compensatory mechanism in the yeast ( Vu et al, 2015 ).…”

“…In addition, the plasma membrane high-affinity Cch1-Mid1 calcium channel (CMC) of C. neoformans promotes cryptococcal survival during exposure to oxidative stress ( Vu et al, 2015 ). Cch1 mutants have decreased survival in J774.1A macrophages, however, C. neoformans deficient in both Cch1 and Mid1 maintain resistance to the ROS, suggesting a compensatory mechanism in the yeast ( Vu et al, 2015 ). C. gattii , however, has been shown to use a novel mechanism in response to ROS.…”