Calcineurin is a eukaryotic Ca2+- and calmodulin-dependent serine/threonine protein phosphatase. It is a heterodimeric protein consisting of a catalytic subunit calcineurin A, which contains an act...

https://www.physiology.org/doi/pdf/10.1152/physrev.2000.80.4.1483

Calcineurin : form and function

We present an analysis of over 1,100 of the approximately 10,000 predicted proteins encoded by the genome sequence of the filamentous fungus Neurospora crassa. Seven major areas of Neurospora genomics and biology are covered. First, the basic features of the genome, including the automated assembly, gene calls, and global gene analyses are summarized. The second section covers components of the centromere and kinetochore complexes, chromatin assembly and modification, and transcription and translation initiation factors. The third area discusses genome defense mechanisms, including repeat induced point mutation, quelling and meiotic silencing, and DNA repair and recombination. In the fourth section, topics relevant to metabolism and transport include extracellular digestion; membrane transporters; aspects of carbon, sulfur, nitrogen, and lipid metabolism; the mitochondrion and energy metabolism; the proteasome; and protein glycosylation, secretion, and endocytosis. Environmental sensing is the focus of the fifth section with a treatment of two-component systems; GTP-binding proteins; mitogen-activated protein, p21-activated, and germinal center kinases; calcium signaling; protein phosphatases; photobiology; circadian rhythms; and heat shock and stress responses. The sixth area of analysis is growth and development; it encompasses cell wall synthesis, proteins important for hyphal polarity, cytoskeletal components, the cyclin/cyclin-dependent kinase machinery, macroconidiation, meiosis, and the sexual cycle. The seventh section covers topics relevant to animal and plant pathogenesis and human disease. The results demonstrate that a large proportion of Neurospora genes do not have homologues in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. The group of unshared genes includes potential new targets for antifungals as well as loci implicated in human and plant physiology and disease.

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Lessons from the Genome Sequence of Neurospora crassa: Tracing the Path from Genomic Blueprint to Multicellular Organism

Summary
The azole antifungal fluconazole possesses only fungistatic activity in Candida albicans and, therefore, this human pathogen is tolerant to this agent. However, tolerance to fluconazole can be inhibited when C. albicans is exposed to fluconazole combined with the immunosuppressive drug cyclosporin A, which is known to inhibit calcineurin activity in yeast. A mutant lacking both alleles of a gene encoding the calcineurin A subunit (CNA) lost viability in the presence of fluconazole, thus making calcineurin essential for fluconazole tolerance. Consistent with this observation, tolerance to fluconazole was modulated by calcium ions or by the expression of a calcineurin A derivative autoactivated by the removal of its C-terminal inhibitory domain. Interestingly, CNA was also essential for tolerance to other antifungal agents (voriconazole, itraconazole, terbinafine, amorolfine) and to several other metabolic inhibitors (caffeine, brefeldin A, mycophenolic acid, fluphenazine) or cell wall-perturbing agents (SDS, calcofluor white, Congo red), thus indicating that the calcineurin pathway plays an important role in the survival of C. albicans in the presence of external growth inhibitors. Several genes, including PMC1, a vacuolar calcium P-type ATPase, were regulated in a calcineurin- and fluconazole-dependent manner. However, PMC1 did not play a direct role in the survival of C. albicans when exposed to fluconazole. In addition to these different properties, calcineurin was found to affect colony morphology in several media known to modulate the C. albicans dimorphic switch. In particular, calcineurin was found to be essential for C. albicans viability in serum-containing media. Finally, calcineurin was found to be necessary for the virulence of C. albicans in a mice model of infection, thus making calcineurin an important element for adequate adaptation to the conditions of the host environment.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-2958.2003.03495.x

Calcineurin A of Candida albicans: involvement in antifungal tolerance, cell morphogenesis and virulence

Publisher Summary The use of the calcineurin inhibitors—namely, FK506 and CsA, together with yeast genetics and the overexpression of calcineurin by transgenic mice, has established the critical roles of calcineurin in the regulation of many cellular processes that are induced by changes in the concentration of intracellular Ca 2+ in response to external signals. None of the physiological roles of calcineurin is better documented than the regulation of gene expression mediated by the broadly distributed NFAT family of transcription factors in mammalian cells. Equally well documented is the role of calcineurin in the regulation of expression of many genes that are under the control of the TCNI/CRZl transcription factor in yeast. Calcineurim was originally identified as a major calmodulin-binding protein in the brain and later shown to be the only Ca 2+ /calmodulin-regulated serine/threonine protein phosphatase. Since then, this enzyme has been shown to be expressed in every tissue and to be highly conserved phylogenetically.

Calcineurin: from structure to function.

Calcineurin is implicated in a myriad of human diseases as well as homeostasis and virulence in several major human pathogenic microorganisms. The fungus Aspergillus fumigatus is a leading cause of infectious death in the rapidly expanding immunocompromised patient population. Current antifungal treatments for invasive aspergillosis are often ineffective, and novel therapeutic approaches are urgently needed. We demonstrate that a mutant of A. fumigatus lacking the calcineurin A (cnaA) catalytic subunit exhibited defective hyphal morphology related to apical extension and polarized growth, which resulted in drastically decreased filamentation. The ΔcnaA mutant lacked the extensive lattice of invading hyphae seen with the wild-type and complemented strains. Sporulation was also affected in the ΔcnaA mutant, including morphological conidial defects with the absence of surface rodlets and the added presence of disjunctors creating long conidial chains. Infection with the ΔcnaA mutant in several distinct animal models with different types of immunosuppression and inoculum delivery led to a profound attenuation of pathogenicity compared to infection with the wild-type and complemented strains. Lung tissue from animals infected with the ΔcnaA mutant showed a complete absence of hyphae, in contrast to tissue from animals infected with the wild-type and complemented strains. Quantitative fungal burden and pulmonary infarct scoring confirmed these findings. Our results support the clinical observation that substantially decreasing fungal growth can prevent disease establishment and decrease mortality. Our findings reveal that calcineurin appears to play a globally conserved role in the virulence of several pathogenic fungi and yet plays specialized roles in each and can be an excellent target for therapeutic intervention.

Calcineurin Controls Growth, Morphology, and Pathogenicity in Aspergillus fumigatus

The function of Neurospora crassa calcineurin was investigated in N. crassa strains transformed with a construct that provides for the inducible expression of antisense RNA for the catalytic subunit of calcineurin (cna-1). Induction of antisense RNA expression was associated with reduced levels of cna-1 mRNA and of immunodetectable CNA1 protein and decreased cal- cineurin enzyme activity, indicating that a conditional reduction of the target function had been achieved in antisense transformants with multiple construct inte- grations. Induction conditions caused growth arrest which indicated that the cna-1 gene is essential for growth of N. crassa. Growth arrest was preceded by an increase in hyphal branching, changes in hyphal mor- phology and concomitant loss of the distinctive tip-high Ca 2+ gradient typical for growing wild-type hyphae. This demonstrates a novel and specific role for cal- cineurin in the precise regulation of apical growth, a common form of cellular proliferation. In vitro inhi- bition of N. crassa calcineurin by the complex of cy- closporin A (CsA) and cyclophilin20, and increased sensitivity of the induced transformants to the cal- cineurin-specific drugs CsA and FK506 imply that the drugs act in N. crassa, as in T-cells and Saccharomyces cerevisiae, by inactivating calcineurin. The finding that exposure of growing wild-type mycelium to these drugs leads to a phenotype very similar to that of the cna-1 antisense mutants is consistent with this idea.

Impairment of calcineurin function in Neurospora crassa reveals its essential role in hyphal growth, morphology and maintenance

The function of Neurospora crassa calcineurin was investigated in N. crassa strains transformed with a construct that provides for the inducible expression of antisense RNA for the catalytic subunit of calcineurin (cna-1). Induction of antisense RNA expression was associated with reduced levels of cna-1 mRNA and of immunodetectable CNA1 protein and decreased calcineurin enzyme activity, indicating that a conditional reduction of the target function had been achieved in antisense transformants with multiple construct integrations. Induction conditions caused growth arrest which indicated that the cna-1 gene is essential for growth of N. crassa. Growth arrest was preceded by an increase in hyphal branching, changes in hyphal morphology and concomitant loss of the distinctive tip-high Ca2+ gradient typical for growing wild-type hyphae. This demonstrates a novel and specific role for calcineurin in the precise regulation of apical growth, a common form of cellular proliferation. In vitro inhibition of N. crassa calcineurin by the complex of cyclosporin A (CsA) and cyclophilin20, and increased sensitivity of the induced transformants to the calcineurin-specific drugs CsA and FK506 imply that the drugs act in N. crassa, as in T-cells and Saccharomyces cerevisiae, by inactivating calcineurin. The finding that exposure of growing wild-type mycelium to these drugs leads to a phenotype very similar to that of the cna-1 antisense mutants is consistent with this idea.

Impairment of calcineurin function in Neurospora crassa reveals its essential role in hyphal growth, morphology and maintenance of the apical Ca2+ gradient

M. Dieminger

Papers

Impairment of calcineurin function in Neurospora crassa reveals its essential role in hyphal growth, morphology and maintenance

Impairment of calcineurin function in Neurospora crassa reveals its essential role in hyphal growth, morphology and maintenance of the apical Ca2+ gradient