Comparison of the Roles of Calcineurin in Physiology and Virulence in Serotype D and Serotype A Strains of Cryptococcus neoformans

Abstract

The calcineurin gene was cloned and disrupted in serotype D strains of Cryptococcus neoformans. Serotype A and serotype D calcineurin mutants were inviable at 37°C and avirulent in mice, whereas only serotype A mutants were cation stress sensitive. Thus, calcineurin plays conserved and divergent roles in serotype A and serotype D strains.Cryptococcus neoformans is an encapsulated basidiomycete that is the most common cause of systemic mycosis in AIDS patients. C. neoformans strains are classified into five serotypes (A, B, C, D, and AD) and two varieties: C. neoformans var. neoformans (serotypes A, D, and AD) and C. neoformans var. gattii (serotypes B and C). Serotype A and serotype D strains exhibit significant variation and may represent distinct varieties that have diverged in ϳ18 million years of evolution (8,9,16,19,25). C. neoformans virulence factors include the capsule (3-5), melanin (22), prototrophy (17), and growth at 37°C (14). The protein phosphatase calcineurin is required for C. neoformans growth at 37°C and virulence (14).Calcineurin is a Ca 2ϩ -calmodulin-activated phosphatase with catalytic and regulatory subunits (10). Calcineurin is the target of the T-cell-specific immunosuppressants cyclosporine (CsA) and tacrolimus (FK506). CsA and FK506 suppress the immune system by inhibiting calcineurin and preventing gene expression during T-cell activation. The antifungal activities of CsA and FK506 are mediated by a similar mechanism involving fungal homologs of calcineurin and the drug-binding proteins cyclophilin A and FKBP12 (1,2,6,7,14,15).Calcineurin has been identified from several fungi and regulates cell cycle progression in Aspergillus nidulans (21), hyphal elongation and growth in Neurospora crassa (11,20), and mating and cytokinesis in Schizosaccharomyces pombe (18,28). In Saccharomyces cerevisiae, calcineurin is required for recovery from pheromone arrest (12, 27) and regulates cation homeostasis and cell wall biosynthesis via the transcription factor Crz1 (1,13,23,24).The calcineurin gene has been identified, sequenced, and disrupted by homologous recombination in C. neoformans serotype A strain H99 (14). Calcineurin is essential for growth at 37°C, virulence in a rabbit model of cryptococcal meningitis, and cation homeostasis (14). Here, we isolated and disrupted the gene encoding the calcineurin A catalytic subunit (CNA1) from the congenic serotype D strains of C. neoformans and compared the functions of calcineurin in serotype A and serotype D strains.Identification, sequence, and disruption of the serotype D calcineurin A CNA1 gene. The CNA1 gene encoding calcineurin A was cloned from the serotype D strain JEC21 by PCR amplification with primers to conserved sequences in the serotype A CNA1 gene. A 1.8-kb CNA1 gene fragment was sequenced, revealing identity to calcineurin genes, and used in Southern blot analysis to show that the CNA1 gene is contained on an 8-kb EagI fragment. The gene was recovered from a size-selected EagI genomic library and sequenced. There were seven amino acid...

Signal Transduction Cascades Regulating Fungal Development and Virulence

“…Calcineurin was found to be essential for growth at 37°C and virulence in both the rabbit model of cryptococcal meningitis and a murine inhalation model. Serotype A mutants are hypersensitive to Na ϩ and Li ϩ compared to the isogenic wild type, suggesting a role for calcineurin analogous to that in S. cerevisiae, in which calcineurin regulates expression of the Na ϩ and Li ϩ efflux pump Pmr2 (61). In serotype D, however, calcineurin does not appear to regulate cation homeostasis (61).…”

“…Calcineurin has been identified in several fungi, including S. cerevisiae, in which it regulates cation homeostasis, cell wall biosynthesis, and recovery from pheromone growth arrest. In C. neoformans, the calcineurin A gene has been identified and disrupted by homologous recombination in both serotype A and D strains (61,217). Calcineurin was found to be essential for growth at 37°C and virulence in both the rabbit model of cryptococcal meningitis and a murine inhalation model.…”

“…One candidate downstream target of the MAP kinase cascade, the STE12␣ homolog, is present only in MAT␣ cells and may play a role in the increased virulence of this mating type in serotype D strains (47,300,320). Finally, the role of calcineurin in both serotypes A and D has been investigated, as well as the potential for calcineurin inhibitors to serve as antifungal compounds (60,61,68,217).…”

Immunosuppresseurs et antifongiques

“…Ce mécanisme a été mis en évi-dence par des expériences de délétion du gène codant pour la sousunité catalytique de la calcineurine [12]. Chez C. neoformans, la voie de la calcineurine permet la croissance des levures à 37°C et contrôle la virulence du pathogène [13,14]. La croissance du mycélium d'Aspergillus fumigatus [15,16] et l'adaptation au stress d'A.…”

Unisexual Reproduction Reverses Muller’s Ratchet

“…We tested whether parental strains with mutations that compromise virulence are able to undergo unisexual reproduction to produce virulent progeny. Studies of C. neoformans have shown that calcineurin is required for virulence in mammalian models (Odom et al 1997;Cruz et al 2000;Fox et al 2001). We injected inocula containing C. neoformans into the G. mellonella larvae to assess the virulence of parental and progeny strains.…”