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.

FIG. 1.

C. neoformans T-DNA insertional mutants with in vitro phenotypes and associated mutated genes. (A) Cells were 10-fold serially diluted and spotted onto YPD, YNB, or l-DOPA agar medium. One YPD plate was exposed to UV irradiation (180 J/m²). Plates were incubated at 30 or 37°C for 2 days or at 22°C for 4 days with l-DOPA. (B) Regions flanking the T-DNA in each strain were identified by inverse PCR and used to determine the disrupted gene by comparison against the C. neoformans genome database. For DNA alignments, the top sequence is from the wild-type strain, with bold letters denoting those nucleotides replaced by the T-DNA insertion event, except in the case of strain ST310F6, which has a duplication of GAT (underlined). Gray font indicates the T-DNA sequence, with italics indicating right- or left-border nucleotides.

FIG. 2.

Random T-DNA insertion points in the C. neoformans genome. The regions flanking the T-DNAs were compared to the serotype A genome sequence (Broad Institute chromosome designation, 8 December 2008) and mapped onto all 14 chromosomes.

FIG. 3.

ENA1 is required for virulence. Mice were inoculated with wild-type C. neoformans strain KN99α (n = 10), ena1 deletion mutants of both mating types (n = 9 for both groups), and two independent ena1 ENA1 (ena1Δ+ENA1) complemented strains (n = 9 for both groups).

FIG. 4.

The mutation of ENA1 causes increased sensitivity to alkaline pH. (A) Cells were 10-fold serially diluted and spotted onto YPD at a pH of 7 or at a pH adjusted to ∼8.5 with KOH, and the cultures were incubated at 30°C for 2 or 3 days, respectively. (B and C) Strains were grown in liquid YPD adjusted to different pHs with Tris base at 22°C, and the optical density at 600 nm (OD 600nm) was measured daily. An optical density of 4 was the maximum reading. (B) Comparative growth rates for all three strains under four pH conditions. (C) Reduced growth of the ena1 mutant at pH 8.6 compared to that of the wild-type and complemented strains.

FIG. 5.

Calcineurin and Ena1 jointly contribute to growth under alkaline conditions. (A) The mutation of CNA1 causes increased sensitivity to alkaline pH. Cells were 10-fold serially diluted and spotted onto YPD at a pH of 7 or at a pH adjusted to ∼8.5 with NaOH, and the cultures were incubated at 30°C for 2 days (pH 7) or 3 days (pH 8.5) or at 37°C for 2 days. (B) ENA1 transcript levels increase with high pH. Strains were grown in regular YPD liquid or YPD liquid adjusted to pH 8.3 with NaOH for a 2-h incubation. Total RNA was extracted from the cells and was probed for ENA1 and actin (ACT1) mRNA levels. (C) The combined mutation of CNA1 and ENA1 causes an additional increase in sensitivity to alkaline pH. Cells were 10-fold serially diluted and spotted onto YPD at a pH of 7 or at a pH adjusted to ∼8.0 with NaOH, and the cultures were incubated at 30°C for 2 days (pH 7) or 3 days (pH 8).