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

Pathogenic 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 (Ca2+) 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 H2O2 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 H2O2 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 Delta 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 H2O2 stress, suggesting the existence of a compensatory mechanism that becomes active in the absence of CMC.