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The Crz1/Sp1 transcription factor of Cryptococcus neoformans is activated by calcineurin and regulates cell wall integrity.

ABSTRACT: Cryptococcus neoformans survives host temperature and regulates cell wall integrity via a calcium-dependent phosphatase, calcineurin. However, downstream effectors of C. neoformans calcineurin are largely unknown. In S. cerevisiae and other fungal species, a calcineurin-dependent transcription factor Crz1, translocates to nuclei upon activation and triggers expression of target genes. We now show that the C. neoformans Crz1 ortholog (Crz1/Sp1), previously identified as a protein kinase C target during starvation, is a bona fide target of calcineurin under non-starvation conditions, during cell wall stress and growth at high temperature. Both the calcineurin-defective mutant, ?cna1, and a CRZ1/SP1 mutant (?crz1) were susceptible to cell wall perturbing agents. Furthermore, expression of the chitin synthase encoding gene, CHS6, was reduced in both mutants. We tracked the subcellular localization of Crz1-GFP in WT C. neoformans and ?cna1 in response to different stimuli, in the presence and absence of the calcineurin inhibitor, FK506. Exposure to elevated temperature (30-37°C vs 25°C) and extracellular calcium caused calcineurin-dependent nuclear accumulation of Crz1-GFP. Unexpectedly, 1M salt and heat shock triggered calcineurin-independent Crz1-GFP sequestration within cytosolic and nuclear puncta. To our knowledge, punctate cytosolic distribution, as opposed to nuclear targeting, is a unique feature of C. neoformans Crz1. We conclude that Crz1 is selectively activated by calcium/calcineurin-dependent and independent signals depending on the environmental conditions.

SUBMITTER: Lev S

PROVIDER: S-EPMC3520850 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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The Crz1/Sp1 transcription factor of Cryptococcus neoformans is activated by calcineurin and regulates cell wall integrity.

Lev Sophie S Desmarini Desmarini D Chayakulkeeree Methee M Sorrell Tania C TC Djordjevic Julianne T JT

PloS one 20121212 12

Cryptococcus neoformans survives host temperature and regulates cell wall integrity via a calcium-dependent phosphatase, calcineurin. However, downstream effectors of C. neoformans calcineurin are largely unknown. In S. cerevisiae and other fungal species, a calcineurin-dependent transcription factor Crz1, translocates to nuclei upon activation and triggers expression of target genes. We now show that the C. neoformans Crz1 ortholog (Crz1/Sp1), previously identified as a protein kinase C target ...[more]

PMID: 23251520

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Project description:Calcineurin is a highly conserved Ca2+/calmodulin-dependent serine/threonine-specific protein phosphatase that orchestrates cellular Ca2+ signaling responses. In Cryptococcus neoformans, calcineurin is activated by multiple stresses including high temperature, and is essential for stress adaptation and virulence. The transcription factor Crz1 is a major calcineurin effector in Saccharomyces cerevisiae and other fungi. Calcineurin dephosphorylates Crz1, thereby enabling Crz1 nuclear translocation and transcription of target genes. Here we show that loss of Crz1 confers phenotypes intermediate between wild-type and calcineurin mutants, and demonstrate that deletion of the calcineurin docking domain results in the inability of Crz1 to translocate into the nucleus under thermal stress. RNA-sequencing revealed 102 genes that are regulated in a calcineurin-Crz1-dependent manner at 37°C. The majority of genes were down-regulated in cna1Δ and crz1Δ mutants, indicating these genes are normally activated by the calcineurin-Crz1 pathway at high temperature. About 58% of calcineurin-Crz1 target genes have unknown functions, while genes with known or predicted functions are involved in cell wall remodeling, calcium transport, and pheromone production. We identified three calcineurin-dependent response element motifs within the promoter regions of calcineurin-Crz1 target genes, and show that Crz1 binding to target gene promoters is increased upon thermal stress in a calcineurin-dependent fashion. Additionally, we found a large set of genes independently regulated by calcineurin, and Crz1 regulates 59 genes independently of calcineurin. Given the intermediate crz1Δ mutant phenotype, and our recent evidence for a calcineurin regulatory network impacting mRNA in P-bodies and stress granules independently of Crz1, calcineurin likely acts on factors beyond Crz1 that govern mRNA expression/stability to operate a branched transcriptional/post-transcriptional stress response network necessary for fungal virulence. Taken together, our findings reveal the core calcineurin-Crz1 stress response cascade is maintained from ascomycetes to a pathogenic basidiomycete fungus, but its output in C. neoformans appears to be adapted to promote fungal virulence.

Project description:Calcineurin is a highly conserved Ca2+/calmodulin-dependent serine/threonine-specific protein phosphatase that orchestrates cellular Ca2+ signaling responses. In Cryptococcus neoformans, calcineurin is activated by multiple stresses including high temperature, and is essential for stress adaptation and virulence. The transcription factor Crz1 is a major calcineurin effector in Saccharomyces cerevisiae and other fungi. Calcineurin dephosphorylates Crz1, thereby enabling Crz1 nuclear translocation and transcription of target genes. Here we show that Crz1 is dephosphorylated by calcineurin, and crz1Δ mutants display phenotypes intermediate between wild-type and calcineurin mutants. RNA-sequencing revealed 102 genes that are regulated in a calcineurin/Crz1-dependent manner at 37°C. 99 genes were down-regulated in cna1Δ and crz1Δ mutants, indicating these genes are normally activated by the calcineurin/Crz1 pathway at high temperature. About 58% of calcineurin-Crz1 target genes have unknown functions, while genes with known or predicted functions are involved in cell wall remodeling, calcium transport, and pheromone production. Surprisingly, only five genes have orthologs known to be calcineurin/Crz1-dependent in S. cerevisiae. 393 genes are independently regulated by calcineurin, and Crz1 regulates 59 genes independently of calcineurin. Taken together, these results indicate that the calcineurin/Crz1-dependent pathway controls a transcriptional circuit that has been extensively rewired in C. neoformans compared to S. cerevisiae. Given the intermediate crz1Δ mutant phenotype, and our recent evidence for a calcineurin regulatory network impacting mRNA in P-bodies and stress granules independently of Crz1, calcineurin likely acts on factors beyond Crz1 that govern mRNA expression/stability to operate a branched transcriptional/post-transcriptional stress response network necessary for fungal virulence. Taken together, our findings reveal the core calcineurin-Crz1 stress response cascade is maintained from ascomycetes to a pathogenic basidiomycete fungus, but its output has been extensively rewired to promote fungal virulence.

Project description:Morphotype switch is a cellular response to external and internal cues. The Cryptococcus neoformans species complex can undergo morphological transitions between the yeast and the hypha form, and such morphological changes profoundly affect cryptococcal interaction with various hosts. Filamentation in Cryptococcus was historically considered a mating response towards pheromone. Recent studies indicate the existence of pheromone-independent signaling pathways but their identity or the effectors remain unknown. Here, we demonstrated that glucosamine stimulated the C. neoformans species complex to undergo self-filamentation. Glucosamine-stimulated filamentation was independent of the key components of the pheromone pathway, which is distinct from pheromone-elicited filamentation. Glucosamine stimulated self-filamentation in H99, a highly virulent serotype A clinical isolate and a widely used reference strain. Through a genetic screen of the deletion sets made in the H99 background, we found that Crz1, a transcription factor downstream of calcineurin, was essential for glucosamine-stimulated filamentation despite its dispensability for pheromone-mediated filamentation. Glucosamine promoted Crz1 translocation from the cytoplasm to the nucleus. Interestingly, multiple components of the high osmolality glycerol response (HOG) pathway, consisting of the phosphorelay system and some of the Hog1 MAPK module, acted as repressors of glucosamine-elicited filamentation through their calcineurin-opposing effect on Crz1's nuclear translocation. Surprisingly, glucosamine-stimulated filamentation did not require Hog1 itself and was distinct from the conventional general stress response. The results demonstrate that Cryptococcus can resort to multiple genetic pathways for morphological transition in response to different stimuli. Given that the filamentous form attenuates cryptococcal virulence and is immune-stimulatory in mammalian models, the findings suggest that morphogenesis is a fertile ground for future investigation into novel means to compromise cryptococcal pathogenesis.

Project description:UnlabelledCryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis of C. neoformans, and biosynthesis and repair of the wall is primarily controlled by the cell wall integrity (CWI) signaling pathway. Previous work has shown that deletion of genes encoding the four major kinases in the CWI signaling pathway, namely, PKC1, BCK1, MKK2, and MPK1 results in severe cell wall phenotypes, sensitivity to a variety of cell wall stressors, and for Mpk1, reduced virulence in a mouse model. Here, we examined the global transcriptional responses to gene deletions of BCK1, MKK2, and MPK1 compared to wild-type cells. We found that over 1,000 genes were differentially expressed in one or more of the deletion strains, with 115 genes differentially expressed in all three strains, many of which have been identified as genes regulated by the cyclic AMP (cAMP)/protein kinase A (PKA) pathway. Biochemical measurements of cAMP levels in the kinase deletion strains revealed significantly less cAMP in all of the deletion strains compared to the wild-type strain. The deletion strains also produced significantly smaller capsules than the wild-type KN99 strain did under capsule-inducing conditions, although the levels of capsule they shed were similar to those shed by the wild type. Finally, addition of exogenous cAMP led to reduced sensitivity to cell wall stress and restored surface capsule to levels near those of wild type. Thus, we have direct evidence of cross talk between the CWI and cAMP/PKA pathways that may have important implications for regulation of cell wall and capsule homeostasis.ImportanceCryptococcus neoformans is a fungal pathogen of immunocompromised people that causes fatal meningitis. The fungal cell wall is essential to viability and pathogenesis of C. neoformans, and biosynthesis and repair of the wall are primarily controlled by the cell wall integrity (CWI) signaling pathway. In this study, we demonstrate that deletion of any of three core kinases in the CWI pathway impacts not only the cell wall but also the amount of surface capsule. Deletion of any of the kinases results in significantly reduced cellular cyclic AMP (cAMP) levels, and addition of exogenous cAMP rescues the capsule defect and some cell wall defects, supporting a direct role for the CWI pathway in regulation of capsule in conjunction with the cAMP/protein kinase A pathway.

Dataset Information

The Crz1/Sp1 transcription factor of Cryptococcus neoformans is activated by calcineurin and regulates cell wall integrity.

Publications

The Crz1/Sp1 transcription factor of Cryptococcus neoformans is activated by calcineurin and regulates cell wall integrity.

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