Cryptococcus neoformans virulence gene discovery through insertional mutagenesis

Abstract

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.

Figures

FIG. 1.

Phenotypes of C. neoformans insertional mutants. (A) Four strains generated by biolistic transformation and three generated by Agrobacterium-mediated transformation with altered production of melanin compared to the wild type (H99) were grown on l-DOPA medium for 4 days at room temperature and were then photographed. (B) Strains generated by biolistic transformation with altered sensitivities to 37°C. Serial dilutions of strains were grown for 2 days at 30 or 37°C on YPD medium. (C) Two strains generated by biolistic transformation with altered sensitivities to nitric oxide compared to a mutant in the FHB1 gene and to strain H99. Strains were grown in 0 or 1 mM DETA-NONOate (NO) overnight in YPD liquid medium. Optical densities were measured at 600 nm with a plate reader, and fold increases in growth were plotted.

FIG. 2.

Nitric oxide-hypersensitive strains fail to express flavohemoglobin (FHB1). (A) PCR analysis of wild-type (H99) and insertional mutant (3E7) strains showing a deletion of approximately 18 kb in strain 3E7. Dots indicate PCR amplicons. Actin (ACT1) was used as a control for DNA amplification of both strains. (B) Transcription of the FHB1 gene in wild-type (H99) and fhb1 mutants (targeted deletion of fhb1 and insertional mutants 3E7 and 3C12) was analyzed by Northern blotting with a radiolabeled probe for the FHB1 gene. A probe for the actin gene (ACT1) was used as a control for RNA loading and transfer.

FIG. 3.

Arrangement of insertion of T-DNA by Agrobacterium into the genomes of two mel− mutants of C. neoformans. Sequences of the T-DNA left and right borders, the mutants, and wild-type H99 genomic DNA were aligned. One T-DNA insertion was in the promoter of the CLC1 gene and the other was in the promoter of the LAC1 gene. Small deletions of DNA-containing sequences similar to the T-DNA borders (e.g., TGGCAGGA in left border and region deleted in At-mel2) are associated with the insertion of T-DNA.

FIG. 4.

Diagram of deletion alleles used to examine gene functioning of insertional mutant 1F2. The arrangement of two genes, one represented by an EST and the other being a MAP kinase (MPK2), was derived from The Institute for Genomic Research C. neoformans database. The MPK2 gene sequence was predicted and may be shorter at the 5′ end than is represented. Strain 1F2 had the plasmid conferring resistance to nourseothricin (NAT) inserted at the arrowhead, and a deletion of <1 kb occurred (dashed line) that was not further defined. Three alleles were used to replace the MPK2, the EST, or both genes with the NAT marker.

FIG. 5.

C. neoformans insertional mutant 1F2 exhibits a bilateral mating defect. Crosses between the wild type and the 1F2 mutant or a mutant strain derived by genetic crosses were cocultured on V8 medium (pH 5) in the dark and were examined 10 days later. (A) Wild-type H99 (MATα) × KN99-5 (MATa); (B) unilateral H99 (MATα) × mutant (Δ) 1F2 MATa progeny (NAT MATa); (C) unilateral 1F2 (NAT MATα) × KN99-5 (MATa); (D) bilateral 1F2 (NAT MATα) × 1F2 MATa (NAT MATa). Bar = 0.5 mm.

FIG. 6.

C. neoformans voltage-gated chloride channel gene CLC1 is required for normal growth under low-iron conditions. The growth of wild-type (H99) and clc1 and clc2 mutant strains of C. neoformans was compared to that of reference wild-type (BY4743) and gef1 mutant strains of S. cerevisiae. Cells were serially diluted and grown on synthetic medium containing 0 or 2 mM iron chelation agent ferrozine (3 days at room temperature) (A) or glucose (2 days at 30°C), glucose plus 3-amino-1,2,4-triazole (3-AT) (10 mM; 2 days at 30°C), or acetate (2%; 4 days at 30°C) as the sole carbon source (B).