Calcium binding protein Ncs1 is calcineurin-regulated in Cryptococcus neoformans and essential for cell division and virulence

Intracellular calcium (Ca2+) is crucial for signal transduction in Cryptococcus neoformans, the major cause of fatal fungal meningitis. The calcineurin pathway is the only Ca2+-requiring signalling cascade implicated in cryptococcal stress adaptation and virulence, with Ca2+-binding mediated by the EF-hand domains of the Ca2+ sensor protein calmodulin. In this study, we identified the cryptococcal ortholog of neuronal calcium sensor-1 (Ncs1) as a member of the EF-hand superfamily. We demonstrated that Ncs1 has a role in Ca2+ homeostasis under stress and non-stress conditions, as the ncs1Δ mutant is sensitive to a high Ca2+ concentration and has an elevated basal Ca2+ level that correlates with increased expression of the Ca2+ transporter genes, CCH1 and MID1. Furthermore, NCS1 expression is induced by Ca2+, with the Ncs1 protein adopting a punctate subcellular distribution. We also demonstrate that, in contrast to Saccharomyces cerevisiae, NCS1 expression in C. neoformans is regulated by the calcineurin pathway via the transcription factor Crz1, as NCS1 expression is reduced by FK506 treatment and CRZ1 deletion. Moreover, the ncs1Δ mutant shares a high temperature and high Ca2+ sensitivity phenotype with the calcineurin and calmodulin mutants (cna1Δ and cam1Δ) and the NCS1 promoter contains two calcineurin/Crz1-dependent response elements (CDRE1). Ncs1-deficency coincided with reduced growth, characterized by delayed bud emergence and aberrant cell division, and hypovirulence in a mouse infection model. In summary, our data shows that Ncs1 plays distinct roles in Ca2+ sensing in C. neoformans despite widespread functional conservation of Ncs1 and other regulators of Ca2+ homeostasis. Importance Cryptococcus neoformans is the major cause of fungal meningitis in HIV infected patients. Several studies have highlighted the important contribution of Ca2+ signalling and homeostasis to the virulence of C. neoformans. Here, we identify the cryptococcal ortholog of neuronal calcium sensor-1 (Ncs1) and demonstrate its role in Ca2+ homeostasis, bud emergence, cell cycle progression and virulence. We also show that Ncs1 function is regulated by the calcineurin/Crz1 signalling cascade. Our work provides evidence of a link between Ca2+ homeostasis and cell cycle progression in C. neoformans.


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Cryptococcus neoformans is a basidiomycetous pathogenic yeast, found mostly 35 in soil and bird droppings (1)(2)(3). This pathogen is the etiological agent of 36 cryptococcosis, which affects mainly immunocompromised individuals. This disease 37 affects more than 220,000 HIV-infected patients per year, resulting in more than 38 180,000 deaths worldwide (3,4). The lung infection is initiated following the 39 inhalation of small desiccated cells or spores. The infection can then spread via the 40 bloodstream to the central nervous system, causing meningoencephalitis, which is 41 the primary cause of death (1,5). To survive within the host environment, C. 42 neoformans produces several virulence determinants, including a polysaccharide 43 capsule, the pigment melanin, secreted enzymes (6-9) and extracellular vesicles (10). 44 C. neoformans survival in the host is only possible due to its ability to grow at 37 °C 45 3 and also aided by its capacity to survive within phagocytic mammalian cells (1, 15). 47 Fungal fitness and survival in the host environment is controlled by numerous 48 signaling pathways including those that are regulated by intracellular Ca 2+ , which is 49 an essential second messenger in eukaryotic cells (16)(17)(18)(19)). An increase in cytosolic 50 Ca 2+ is monitored by Ca 2+ sensor proteins that, upon binding to Ca 2+ , change their  Given that high levels of cellular Ca 2+ can be toxic, Ca 2+ homeostasis is strictly 63 regulated by several proteins acting as transporters, channels or pumps (28). In C. 64 neoformans, these proteins include Cch1, a Ca 2+ voltage-gated channel essential for 65 virulence and Mid1, a stretch-activated Ca 2+ -channel, both found in the plasma 66 membrane (19,29). Other cryptococcal calcium transporters that also promote 67 4 virulence include the Ca 2+-ATPase, EcaI, found in sarcoplasmic/endoplasmic 68 reticulum, the H + / Ca 2+ exchanger protein Vcx1 and the Ca 2+ ATPase Pmc1, both 69 localized on vacuolar membranes and responsible for Ca 2+ storage (30-33). Pmc1 is 70 also required for C. neoformans transmigration through the blood-brain barrier 71 (BBB), proving that Pmc1-regulated Ca 2+ homeostasis is crucial for disease 72 progression (33). 73 Despite the importance of Ca 2+ homeostasis-related proteins in fungal cell 74 fitness and virulence, with the exception of calmodulin, little is known about the 75 function of other calcium binding proteins (CBPs) that act as Ca 2+ sensors in C. 76 neoformans. One such protein is the neuronal calcium sensor 1 (Ncs1). Ncs-1 77 orthologs in other fungi have roles in cell growth and viability, tolerance to Ca 2+ (34-78 41), membrane sterol distribution and expression of Ca 2+ transporter genes (41). 79 Here, we identify and characterize the Ncs1 ortholog in C. neoformans Ca 2+ -calcineurin pathway, such as growth in the presence of cell wall perturbing 123 agents (Calcofluor white and Congo red) and osmotic stress (1 M NaCl) were 124 evaluated in the ncs1∆ null mutant, with no effect observed ( Fig S2). 125 We also evaluated whether the level of free intracellular Ca 2+ in C. neoformans 126 is affected in the absence of Ncs1. Relative to the WT strain, the ncs1∆ mutant had a 127 higher basal level of free cytosolic Ca 2+ , which was reduced to WT levels in the 128 ncs1∆::NCS1 strain. This high Ca 2+ level phenotype was shared with that observed for 129 the cna1∆ and cam1∆ mutant strains ( Fig 3A) (Fig 3B). However, expression of both genes increased by approximately 3-fold in the 140 ncs1∆ mutant strain following growth in the presence of Ca 2+ (Fig 3B). This suggests 141 that Cch1 and Mid1 could be the potential source of the extra Ca 2+ in the ncs1∆ mutant, 142 since they import Ca 2+ to the cytosol (29,47). We also generated a mid1∆ncs1∆ double 143 mutant in C. neoformans to evaluate if calcium sensitivity would be restored.  We also C-terminally tagged Ncs1 with GFP (NCS1::GFP) to assess Ncs1 148 subcellular localization. Faint Ncs1 fluorescence was observed when the strain was 149 cultured in the absence of Ca 2+ , however it was higher than that observed for the non-   7A), suggesting that Ncs1 could play a role in cell cycle progression. We therefore 203 investigated the growth defect further by determining the time it took for buds to 204 emerge using time-lapse microscopy ( Fig. 7B and Movies S1 and S2). Given that the 205 mutant was severely attenuated in growth when cultured in DMEM or exposed to 206 mouse serum, we chose YPD medium for this analysis, as it is a richer medium where 207 mutant growth is not as compromised. To avoid bias due to lack of synchronization, 208 we only measured the time of bud emergence in cells after the bud of the first 209 daughter cell had separated from the mother or, in the case of the mutant cells, where 210 progeny did not detach from mother cell, after the second bud emergence. The results 211 demonstrate that it took ~70 min for buds to emerge in the WT cells and more than 212 140 min for buds to emerge in isolated and clumped ncs1Δ mutant cells (Fig. 7C).

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Furthermore, buds were slow to be released in some ncs1Δ mutant cells, resulting in 214 more extensive cell clumping.

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As cell division is linked to the cell cycle, we evaluated whether cells lacking 216 NCS1 displayed defects in cell cycle regulation by measuring the levels of two 217 transcripts associated with different stages of the cell cycle: the G1 cyclin encoded by 218 CNL1 (50) and the S phase DNA replication licensing factor encoded by MCM2 (51,52). 219 We also measured the transcript levels of the G protein coupled receptor encoded by 220 GPA2, which displays oscillatory expression during the cell cycle (51,52). All three 221 genes were upregulated in the ncs1∆ strain compared to WT after 4 h of growth in 222 YPD (Fig. 7D), reinforcing that cell cycle progression is altered in the ncs1Δ mutant 223 strain.

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Our results indicate that NCS1 expression in C. neoformans is regulated by Ca 2+ 226 and the calcineurin/Crz1 pathway and corroborate findings on the Ncs1 ortholog in 227 fission yeast (35). In contrast to our conclusions and those made in studies using S. 228 pombe, the S. cerevisiae Ncs1 ortholog, Frq1, was found to be essential for viability  Virulence assay. Virulence assays were performed as previously described (66).   Time-lapse microscopy. Cellular division was followed using confocal microscopy.

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The experimental design was performed as already described (50), with few