Dataset Information

Identification of ENA1 as a virulence gene of the human pathogenic fungus Cryptococcus neoformans through signature-tagged insertional mutagenesis.

ABSTRACT: A library of more than 4,500 signature-tagged insertion mutants of the human pathogenic fungus Cryptococcus neoformans was generated, and a subset was screened in a murine inhalation model to identify genes required for virulence. New genes that regulate aspects of C. neoformans virulence were also identified by screening the entire library for in vitro phenotypes related to the ability to cause disease, including melanin production, growth at high temperature, and growth under conditions of nutrient limitation. A screen of 10% of the strain collection in mice identified an avirulent mutant strain with an insertion in the ENA1 gene, which is predicted to encode a fungus-specific sodium or potassium P-type ATPase. The results of the deletion of the gene and complementation experiments confirmed its key role in mammalian virulence. ena1 mutant strains exhibited no change in sensitivity to high salt concentrations but were sensitive to alkaline pH conditions, providing evidence that the fungus may have to survive at elevated pH during infection of the mammalian host. The mutation of the well-characterized virulence factor calcineurin (CNA1) also rendered C. neoformans strains sensitive to elevated pH. ENA1 transcripts in wild-type and cna1 mutant strains were upregulated in response to high pH, and cna1 ena1 double mutant strains exhibited increased sensitivity to elevated pH, indicating that at least two pathways in the fungus mediate survival under alkaline conditions. Signature-tagged mutagenesis is an effective strategy for the discovery of new virulence genes in fungal pathogens of animals.

SUBMITTER: Idnurm A

PROVIDER: S-EPMC2653249 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Identification of ENA1 as a virulence gene of the human pathogenic fungus Cryptococcus neoformans through signature-tagged insertional mutagenesis.

Idnurm Alexander A Walton Felicia J FJ Floyd Anna A Reedy Jennifer L JL Heitman Joseph J

Eukaryotic cell 20090116 3

A library of more than 4,500 signature-tagged insertion mutants of the human pathogenic fungus Cryptococcus neoformans was generated, and a subset was screened in a murine inhalation model to identify genes required for virulence. New genes that regulate aspects of C. neoformans virulence were also identified by screening the entire library for in vitro phenotypes related to the ability to cause disease, including melanin production, growth at high temperature, and growth under conditions of nut ...[more]

PMID: 19151325

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Project description: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.

Project description:The Cryptococcus complex consists of at least seven evolutionary divergent lineages and causes ?200,000 fatal human infections each year worldwide. The dominant lineage is Cryptococcus neoformans which consists of three haploid clades VNI, VNII, and VNB, their haploid hybrids, and various diploids derived from intra- and inter-clade mating events. In this study, we analyzed the mitogenomes of 184 strains of C. neoformans. Our analyses revealed that all 184 mitogenomes contained the same 15 protein-coding genes in the same gene order. However, their mitogenome sizes varied between 24,740 and 31,327 bp, primarily due to differences in the number and size of mitochondrial introns. Twelve nucleotide sites within five mitochondrial genes were found to contain introns in at least one of the 184 strains, ranging from 2 to 7 introns within each mitogenome. The concatenated mitochondrial exon sequences of the 15 protein-coding genes and two rRNA genes showed that VNI, VNII, and VNB strains were separated into distinct clades or sub-clades, largely consistent with results based on nuclear genome SNPs. However, several novel findings were observed. First, one strain of the VNB clade contained mitogenome exon sequences identical to the main VNI mitogenome type but was distant to other VNB mitogenomes. Second, hybrids among clades VNI, VNII, and VNB identified based on their nuclear genome SNPs contained mitogenomes from different clades, with evidence of their mitogenomes inherited from either the MAT a or the MAT ? parents. Third, the eight diploid VNB (C. neoformans) × VNIV (C. deneoformans) hybrids contained recombinant mitogenomes. Fourth, analyses of intron distribution and the paired exon-intron phylogenies for each of the 12 exon-intron pairs suggested frequent gains and losses of mitochondrial introns during the evolution of C. neoformans. The combined mitogenome exon-based phylogeny and intron distributions suggested that clades VNI, VNII and VNB could be further divided into sub-clades. Together, our results revealed a dynamic evolution of mitochondrial genomes in this important human fungal pathogen.

Dataset Information

Identification of ENA1 as a virulence gene of the human pathogenic fungus Cryptococcus neoformans through signature-tagged insertional mutagenesis.

Publications

Identification of ENA1 as a virulence gene of the human pathogenic fungus Cryptococcus neoformans through signature-tagged insertional mutagenesis.

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