Cryptococcus neoformans Virulence Gene Discovery through Insertional Mutagenesis

• DOI: 10.1128/ec.3.2.420-429.2004
• Publication Date: 2004-04-09T20:03:08Z
• Authors: Alexander Idnurm; Jennifer L. Reedy; Jesse C. Nussbaum; Joseph Heitman
• Keywords: Insertional mutagenesis

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