Molecular Analysis of the Cryptococcus neoformans ADE2 Gene, a Selectable Marker for Transformation and Gene Disruption

Sudarshan, Shruthi; Rc, Davidson; Heitman, Joseph; Ja, Alspaugh

doi:10.1006/fgbi.1999.1126

Cited by 38 publications

(34 citation statements)

References 40 publications

(28 reference statements)

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“…The ADE2 selectable marker was inserted into a unique BglII site within the PKA1 gene, and the resulting pka1::ADE2 disruption allele was used to replace the wild-type PKA1 gene in the ade2 serotype A strain M001 by biolistic transformation (70). Five pka1 mutant strains were identified from 100 Ade ϩ transformants.…”

Section: Resultsmentioning

confidence: 99%

“…A 410-bp PCR product was cloned, sequenced, and used as a probe to isolate a 12-kb HindIII fragment containing the PKA1 gene from a size-selected genomic library of the serotype A strain H99. The ADE2 gene (2.5-kb BamHI fragment) was inserted at a BglII site in the PKA1 gene and biolistically transformed into the ade2 strain M001 (70). pka1::ADE2 mutants were identified by PCR and confirmed by Southern blot.…”

Section: Methodsmentioning

confidence: 99%

“…2B). The ade2 mutant host strain M001 was originally generated by UV irradiation (63,70) and may differ in virulence potential from the congenic prototrophic parental strain H99. Increased PKA in the pka1ϩPKA1 reconstituted strain may contribute to enhance virulence beyond the wild-type level.…”

Section: Vol 21 2001mentioning

confidence: 99%

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Cyclic AMP-Dependent Protein Kinase Controls Virulence of the Fungal Pathogen Cryptococcus neoformans

D’Souza¹,

Alspaugh²,

Yue³

et al. 2001

Molecular and Cellular Biology

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315

392

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Cryptococcus neoformans is an opportunistic fungal pathogen that infects the human central nervous system. This pathogen elaborates two specialized virulence factors: the antioxidant melanin and an antiphagocytic immunosuppressive polysaccharide capsule. A signaling cascade controlling mating and virulence was identified. The PKA1 gene encoding the major cyclic AMP (cAMP)-dependent protein kinase catalytic subunit was identified and disrupted. pka1 mutant strains were sterile, failed to produce melanin or capsule, and were avirulent. The PKR1 gene encoding the protein kinase A (PKA) regulatory subunit was also identified and disrupted. pkr1 mutant strains overproduced capsule and were hypervirulent in animal models of cryptococcosis. pkr1 pka1 double mutant strains exhibited phenotypes similar to that of pka1 mutants, providing epistasis evidence that the Pka1 catalytic subunit functions downstream of the Pkr1 regulatory subunit. The PKA pathway was also shown to function downstream of the G␣ protein Gpa1 and to regulate cAMP production by feedback inhibition. These findings define a G␣ protein-cAMP-PKA signaling pathway regulating differentiation and virulence of a human fungal pathogen.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Cyclic AMP-Dependent Protein Kinase Controls Virulence of the Fungal Pathogen Cryptococcus neoformans

D’Souza¹,

Alspaugh²,

Yue³

et al. 2001

Molecular and Cellular Biology

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315

392

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“…Importantly, C. neoformans is genetically tractable, with a well-characterized sexual cycle involving mating between two haploid mating types, MATa and MATα (Alspaugh et al, 2000b ;KwonChung, 1975KwonChung, , 1976. Efficient transformation and genedisruption protocols have been developed along with auxotrophic and dominant selectable markers and R. C. Davidson and others overexpression plasmids (Davidson et al, 2000 ;Edman & Kwon-Chung, 1990 ;Hua et al, 2000 ;Sudarshan et al, 1999 ;Toffaletti et al, 1993). Furthermore, genetic analysis has been facilitated by the recent characterization of a stable diploid state, which allows identification and analysis of essential genes .…”

Section: Introductionmentioning

confidence: 99%

A PCR-based strategy to generate integrative targeting alleles with large regions of homology

et al. 2002

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Cryptococcus neoformans is an opportunistic fungal pathogen with a defined sexual cycle for which genetic and molecular techniques are well developed. The entire genome sequence of one C. neoformans strain is nearing completion. The efficient use of this sequence is dependent upon the development of methods to perform more rapid genetic analysis including gene-disruption techniques. A modified PCR overlap technique to generate targeting constructs for gene disruption that contain large regions of gene homology is described. This technique was used to disrupt or delete more than a dozen genes with efficiencies comparable to those previously reported using cloning technology to generate targeting constructs. Moreover, it is shown that disruptions can be made using this technique in a variety of strain backgrounds, including the pathogenic serotype A isolate H99 and recently characterized stable diploid strains. In combination with the availability of the complete genomic sequence, this gene-disruption technique should pave the way for higher throughput genetic analysis of this important pathogenic fungus.

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“…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).…”

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confidence: 99%

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

Cruz

Sia

Olson³

et al. 2000

Infect Immun

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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...

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Molecular Analysis of the Cryptococcus neoformans ADE2 Gene, a Selectable Marker for Transformation and Gene Disruption

Cited by 38 publications

References 40 publications

Cyclic AMP-Dependent Protein Kinase Controls Virulence of the Fungal Pathogen Cryptococcus neoformans

Cyclic AMP-Dependent Protein Kinase Controls Virulence of the Fungal Pathogen Cryptococcus neoformans

A PCR-based strategy to generate integrative targeting alleles with large regions of homology

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

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