Cryptococcus spp. cause life-threatening fungal infection of the central nervous system (CNS), predominantly in patients with a compromised immune system. Why Cryptococcus neoformans has this remarkable tropism for the CNS is not clear. Recent research on cerebral pathogenesis of C. neoformans revealed a predominantly transcellular migration of cryptococci across the brain endothelium; however, the identities of key fungal virulence factors that function specifically to invade the CNS remain unresolved. Here we found that a novel, secreted metalloprotease (Mpr1) that we identified in the extracellular proteome of C. neoformans IMPORTANCE Cryptococcus neoformans is a medically relevant fungal pathogen causing significant morbidity and mortality, particularly in immunocompromised individuals. An intriguing feature is its strong neurotropism, and consequently the hallmark of cryptococcal disease is a brain infection, cryptococcal meningoencephalitis. For C. neoformans to penetrate the central nervous system (CNS), it first breaches the blood-brain barrier via a transcellular pathway; however, the identities of fungal factors required for this transmigration remain largely unknown. In an effort to identify extracellular fungal proteins that could mediate interactions with the brain endothelium, we undertook a proteomic analysis of the extracellular proteome and identified a secreted metalloprotease (Mpr1) belonging to the M36 class of fungalysins. Here we found that Mpr1 promotes migration of C. neoformans across the brain endothelium and into the CNS by facilitating attachment of cryptococci to the endothelium surface, thus underscoring the critical role of M36 proteases in fungal pathogenesis.

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Here we show that the C-terminal tail of Mid1 is a modulatory region that impinges on Cch1 channel activity directly and mediates the trafficking of Mid1 to the plasma membrane. This region consists of the last 24 residues of Mid1, and the functional expression of Mid1 in a human embryonic cell line (HEK293) and in C. neoformans is dependent on this domain. Substitutions of arginine (R619A) or cysteine (C621A) in the modulatory region failed to target Mid1 to the plasma membrane and prevented CMC activity. Interestingly, loss of a predicted protein kinase C (PKC)-phosphorylated serine residue (S605A) had no effect on Mid1 trafficking but did alter the kinetics of Cch1 channel activity. Thus, establishment of Ca 2؉ homeostasis in C. neoformans is dependent on a modulatory domain of Mid1.

Some genes cannot be cloned by conventional methods because in most cases the genes or gene products are toxic to E. coli. CCH1 is a high-affinity Ca 2+ channel present in the plasma membrane of Cryptococcus neoformans and other fungi. Like many toxic genes, the molecular cloning of CCH1 has been a major challenge and consequently direct studies of CCH1 channel activity in heterologous expression systems have been impossible. We devised a straightforward approach that resulted in the molecular cloning and functional expression of CCH1 by exploiting homologous recombination both in vitro and in vivo. This approach precluded the standard enzyme digestion-mediated ligation reactions and the subsequent isolation of plasmids from E. coli. The shuttle plasmid carrying CCH1-GFP, which was prepared in vitro and propagated in yeast, was successfully expressed in a mammalian cell line (HEK293). CCH1 transcripts were detected only in HEK293 cells transfected with the plasmid DNA. Fluorescence microscopy studies revealed the expression of CCH1-GFP fusion protein on the cell surface of HEK293 cells, similar to the localization pattern of a wellcharacterized plasma membrane-associated K + channel. This approach will be particularly useful for genes that encode ion channels and transporters that cannot be cloned by conventional techniques requiring E. coli.

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 ...