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Cryptococcus neoformans virulence gene discovery through insertional mutagenesis.

Publication ,  Journal Article
Idnurm, A; Reedy, JL; Nussbaum, JC; Heitman, J
Published in: Eukaryot Cell
April 2004

Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37 degrees C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37 degrees C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu(+)-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their contributions to fungal physiology. Our findings demonstrate that both insertional mutagenesis methods can be applied to gene identification, but Agrobacterium-mediated transformation is more efficient and generates exclusively stable insertion mutations.

Duke Scholars

Published In

Eukaryot Cell

DOI

ISSN

1535-9778

Publication Date

April 2004

Volume

3

Issue

2

Start / End Page

420 / 429

Location

United States

Related Subject Headings

  • Virulence Factors
  • Streptothricins
  • Promoter Regions, Genetic
  • Phenotype
  • Nitric Oxide
  • Mutation
  • Mutagenesis, Insertional
  • Molecular Sequence Data
  • Mitogen-Activated Protein Kinases
  • Microbiology
 

Citation

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Idnurm, A., Reedy, J. L., Nussbaum, J. C., & Heitman, J. (2004). Cryptococcus neoformans virulence gene discovery through insertional mutagenesis. Eukaryot Cell, 3(2), 420–429. https://doi.org/10.1128/EC.3.2.420-429.2004
Idnurm, Alexander, Jennifer L. Reedy, Jesse C. Nussbaum, and Joseph Heitman. “Cryptococcus neoformans virulence gene discovery through insertional mutagenesis.Eukaryot Cell 3, no. 2 (April 2004): 420–29. https://doi.org/10.1128/EC.3.2.420-429.2004.
Idnurm A, Reedy JL, Nussbaum JC, Heitman J. Cryptococcus neoformans virulence gene discovery through insertional mutagenesis. Eukaryot Cell. 2004 Apr;3(2):420–9.
Idnurm, Alexander, et al. “Cryptococcus neoformans virulence gene discovery through insertional mutagenesis.Eukaryot Cell, vol. 3, no. 2, Apr. 2004, pp. 420–29. Pubmed, doi:10.1128/EC.3.2.420-429.2004.
Idnurm A, Reedy JL, Nussbaum JC, Heitman J. Cryptococcus neoformans virulence gene discovery through insertional mutagenesis. Eukaryot Cell. 2004 Apr;3(2):420–429.

Published In

Eukaryot Cell

DOI

ISSN

1535-9778

Publication Date

April 2004

Volume

3

Issue

2

Start / End Page

420 / 429

Location

United States

Related Subject Headings

  • Virulence Factors
  • Streptothricins
  • Promoter Regions, Genetic
  • Phenotype
  • Nitric Oxide
  • Mutation
  • Mutagenesis, Insertional
  • Molecular Sequence Data
  • Mitogen-Activated Protein Kinases
  • Microbiology