Differences between Cryptococcus neoformans and Cryptococcus gattii in the Molecular Mechanisms Governing Utilization of D-Amino Acids as the Sole Nitrogen Source

Abstract

The ability to grow on media containing certain D-amino acids as a sole nitrogen source is widely utilized to differentiate Cryptococcus gattii from C. neoformans. We used the C. neoformans H99 and C. gattii R265 strains to dissect the mechanisms of D-amino acids utilization. We identified three putative D-amino acid oxidase (DAO) genes in both strains and showed that each DAO gene plays different roles in D-amino acid utilization in each strain. Deletion of DAO2 retarded growth of R265 on eleven D-amino acids suggesting its prominent role on D-amino acid assimilation in R265. All three R265 DAO genes contributed to growth on D-Asn and D-Asp. DAO3 was required for growth and detoxification of D-Glu by both R265 and H99. Although growth of H99 on most D-amino acids was poor, deletion of DAO1 or DAO3 further exacerbated it on four D-amino acids. Overexpression of DAO2 or DAO3 enabled H99 to grow robustly on several D-amino acids suggesting that expression levels of the native DAO genes in H99 were insufficient for growth on D-amino acids. Replacing the H99 DAO2 gene with a single copy of the R265 DAO2 gene also enabled its utilization of several D-amino acids. Results of gene and promoter swaps of the DAO2 genes suggested that enzymatic activity of Dao2 in H99 might be lower compared to the R265 strain. A reduction in virulence was only observed when all DAO genes were deleted in R265 but not in H99 indicating a pathobiologically exclusive role of the DAO genes in R265. These results suggest that C. neoformans and C. gattii divergently evolved in D-amino acid utilization influenced by their major ecological niches.

Fig 1. DAO genes are important for growth on D-amino acids.

(A) Phenotype of R265 daoΔ mutants. (B) CnDAO1 and CnDAO3 play different roles for growth on D-amino acids. Three-fold serial dilutions of each indicated strain were spotted on indicated medium and incubated at 30°C. The strains used in the experiments are given on the left. Two independent deletants of each DAO gene from H99 were assayed. Pictures were taken after incubation for 3 days, 6 days, 10 days or 14 days as indicated. The experiments were repeated twice and representative figures are shown.

Fig 2. DAO genes are important for detoxification of D-amino acids.

Three-fold serial dilutions of each strain were spotted onto YNB medium containing 2% glucose and 10 mM ammonium sulfate supplemented with or without 100 mM D- or L-amino acids. Plates were incubated at 30°C for 2 days and photographed. The strains used in the experiments are given on the left.

Fig 3. Expression of DAO genes is induced by certain D-amino acids.

(A) CgDAO2 expression increases in the presence of D-Ala or D-Pro. YPD grown cells (Y) were washed and transferred to YNB medium containing 10 mM D-Ala, D-Pro or ammonium sulfate (N) for the indicated hours. Total RNA (5 μg) was subjected to northern blot analysis using a CgDAO2 probe. Actin served as loading control. (B and C) Expression profiles of R265 and H99 DAO genes in various D-amino acids. YPD grown cells were washed and transferred to YNB medium containing 10mM of the indicated D-amino acids or ammonium sulfate for 2 h. Northern blots were hybridized with the indicated DAO probes. Signals of each DAO gene were normalized to that of the ACTIN gene and expressed as the relative amount to R265 or H99 RNA from the ammonium sulfate grown cultures. The experiments were repeated three times and the error bar represents standard deviation.

Fig 4. Some DAO genes are functionally interchangeable when overexpressed.

(A) Overexpression of CnDAO2 and CnDAO3 complements the growth deficiency of Cgdao2Δ on three D-amino acids. Strain Cgdao2Δ was transformed with the overexpression construct of the indicated DAO gene. The resulting strains were spotted onto YNB medium containing ammonium sulfate or the indicated D-amino acids and incubated at 30°C. Pictures were taken after 3 days (NH₄) or 5 days (D-Ala, D-Pro and D-Met) of incubation. (B) H99 grows robustly on three D-amino acids when DAO genes are overexpressed. H99 was transformed with the overexpression construct of indicated DAO gene. Spot assays were performed as in (A). (C) Overexpression of every DAO gene complements the growth deficiency of Cndao3Δ. Cndao3Δ and Cgdao3Δ were transformed with each DAO overexpression constructs. The resulting transformants were tested for the ability to grow on D-Glu and the results are summarized. Lighted color letters indicate the original mutants that grow poorly on D-Glu. Dark color letters indicate the original mutants that have no phenotype on D-Glu. Arrow line indicates that overexpression of indicated DAO genes complement the mutant phenotype on D-Glu. (D) Complementation of the growth deficiency of dao mutants on D-Asp by overexpressing DAO genes. The dao mutants showing growth defect on D-Asp were transformed with DAO overexpression constructs. The resulting transformants were tested for the ability to grow on D-D-Asp. Symbols used in the figure are the same as in (C).

Fig 5. Gene and promoter swap between CnDAO2 and CgDAO2.

(A) Diagram of the constructs. Cndao2Δ and Cgdao2Δ were transformed with the indicated constructs. The names of the resulting strains are listed on the left. Dotted line indicates the chromosomal regions flanking the deleted locus. Crosses indicate the crossing over event at the homologous regions. The symbols Act(p) = Actin promoter; HYG = hygromycin resistance gene; NEO = neomycin resistance gene; H2 = CnDAO2 without the promoter; R2 = CgDAO2 without the promoter; H2(f) and R2(f) = flanking region of CnDAO2 and CgDAO2 respectively; H2(p) and R2(p) = promoter of CnDAO2 and CgDAO2 respectively. (B) Spot assay of the gene swapped strains. Approximately 600 cells were spotted on D-Ala and the plates were incubated at 30°C for 11 days and photographed. (C) Relative RNA levels. Log phase cells of the indicated strains were transferred to YNB medium containing 10 mM ammonium sulfate or D-Ala for 2 h and the RNA was isolated. The relative mRNA levels were determined by quantitative RT-PCR. Data were normalized with ACTIN levels and expressed as the relative RNA levels of H99 (upper panel) or R265 (lower panel) grown in ammonium sulfate. The experiments were repeated three times and the error bars represent the standard deviation of three technical repeats.

Fig 6. Virulence of the triple dao deletant of R265 is reduced.

(A and B) BALB/c female mice (5 per group) were infected with the indicated strains either by intravenous injection (IV) or intrapharyngeal aspiration (IPh) and the mortality was monitored. C1624: Cgdao1ΔCgdao2ΔCgdao3Δ triple deletant; C1645: Cgdao1Cgdao3Δ double deletant (derived from C1624 by reconstituting CgDAO2). The experiments were repeated twice and the representative data are shown.