Identification And Functional Studies Of Genes Related To Calcium Sensitivity In Candida Albicans

Candida albicans is a common opportunistic fungal pathogen of a diploid in nature.In recent years,calcium signaling pathway has been the focus of extensive studies due to its essential role in the response of fungal cells to environment stresses.Through a genetic screen using the gene replacement and conditional expression(GRACE)library,we have identified a total of 21 genes whose inactivation renders C.albicans cells sensitive to high levels of extracellular calcium.In addition to the calcium signaling pathway,these 21 genes are also involved in tricarboxylic acid cycle,cell wall integrity pathway,cytokinesis and intracellular pH homeostasis.Calcium sensitivity due to inactivation of nine of these genes can be partially or completely suppressed by cyclosporine A,an inhibitor of calcineurin.Therefore,the calcium sensitivity of nearly a half of these 21 mutations is at least partially due to the activation of the calcium signaling.To further explore the calcium-activated and Crz1-regulated genes,we have constructed the crz1/crz1 inactivation mutant through a novel CRISPR(Clusters of regularly interspaced short palindromic repeats)/Cas9 method.RNA sequencing shows that a total of 219 genes are upregulated and 59 genes are down-regulated by Crz1 in response to calcium stress.Karababa et al.(2006)previously identified 65 calcium-activated and Crz1-dependent genes through DNA microarray,with 40 of them overlapping with our identified 219 genes.Through analysis of promoters of these 40 common genes,we have identified a new potential Crz1 bingding motif [GGAGGC(A/G)(C/G)(A/T)G],which is different from the one [G(C/T)GGT] revelaed in the previous study by Karababa et al.(2006)but similar to the Saccharomyces cerevisiae ScCrz1 bingding motif [GNGGC(G/T)CA].Electrophoretic mobility shift assay indicates that CaCrz1 binds in vitro to both the 5'-G(-375)GGCT-3' and the 5'-T(-333)GAGGCGTTG-3' motifs in the promoter of one of the target genes,UTR2.Chromatin immunoprecipitation analysis demonstrates that CaCrz1 binds in vivo to the promoter region containing these two motifs in the UTR2 promoter.Like the C.albicans plasma membrane protein Rch1 that is the negative regulator of calcium uptake and enriched at the bud neck between the mother cell and the daughter cell,the protein kinase Hsl1 related to the control of cell cycle was shown to localize to the bud neck in a previous study.Interestingly,our GRACE screen has revealed that HSL1 is related to calcium sensitivity.Therefore,we constructed its hsl1/hsl1 inactivation mutant through the CRISPR method.We show that the hsl1/hsl1 inactivation mutant is not only sensitive to high levels of extracellular calcium but also to drugs causing cell wall and cell membrane stresses.Taken together,all results in this study provide a basis for our further and comprehensive understanding of the regulation of intracellular calcium homeostasis in C.albicans and important clues for development of antifungal drug targets.