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

<div><p>The ability to grow on media containing certain D-amino acids as a sole nitrogen source is widely utilized to differentiate <i>Cryptococcus gattii</i> from <i>C</i>. <i>neoformans</i>. We used the <i>C</i>. <i>neoformans</i> H99 and <i>C</i>. <i>gattii</i> R265 strains to dissect the mechanisms of D-amino acids utilization. We identified three putative D-amino acid oxidase (<i>DAO</i>) genes in both strains and showed that each <i>DAO</i> gene plays different roles in D-amino acid utilization in each strain. Deletion of <i>DAO2</i> retarded growth of R265 on eleven D-amino acids suggesting its prominent role on D-amino acid assimilation in R265. All three R265 <i>DAO</i> genes contributed to growth on D-Asn and D-Asp. <i>DAO3</i> 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 <i>DAO1</i> or <i>DAO3</i> further exacerbated it on four D-amino acids. Overexpression of <i>DAO2</i> or <i>DAO3</i> enabled H99 to grow robustly on several D-amino acids suggesting that expression levels of the native <i>DAO</i> genes in H99 were insufficient for growth on D-amino acids. Replacing the H99 <i>DAO2</i> gene with a single copy of the R265 <i>DAO2</i> gene also enabled its utilization of several D-amino acids. Results of gene and promoter swaps of the <i>DAO2</i> 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 <i>DAO</i> genes were deleted in R265 but not in H99 indicating a pathobiologically exclusive role of the <i>DAO</i> genes in R265. These results suggest that <i>C</i>. <i>neoformans</i> and <i>C</i>. <i>gattii</i> divergently evolved in D-amino acid utilization influenced by their major ecological niches.</p></div

Expression of <i>DAO</i> genes is induced by certain D-amino acids.

<p>(A) <i>CgDAO2</i> 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 <i>CgDAO2</i> probe. Actin served as loading control. (B and C) Expression profiles of R265 and H99 <i>DAO</i> 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 <i>DAO</i> probes. Signals of each <i>DAO</i> gene were normalized to that of the <i>ACTIN</i> 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.</p

Virulence of the triple <i>dao</i> deletant of R265 is reduced.

<p>(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: <i>Cgdao1ΔCgdao2ΔCgdao3Δ</i> triple deletant; C1645: <i>Cgdao1Cgdao3Δ</i> double deletant (derived from C1624 by reconstituting <i>CgDAO2</i>). The experiments were repeated twice and the representative data are shown.</p

Summary of growth phenotype associated with deletion of <i>DAO</i> genes.

<p>^a All <i>dao</i> mutants of R265 and H99 grew normally on L-amino acids.</p><p>^b No phenotype.</p><p>^c Growth was only slightly reduced compared to the wild-type.</p><p>^d No D, L-isomers.</p><p>^e Triple deletant of R265 displayed growth defect on D-amino acid.</p><p>Summary of growth phenotype associated with deletion of <i>DAO</i> genes.</p

<i>DAO</i> genes are important for detoxification of D-amino acids.

<p>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.</p

Substrate specificity of Dao enzymes.

<p>^a Substrate specificity of CgDao enzymes (nM H₂O₂/ug crude extract) was compared to the growth phenotypes of <i>Cgdao</i> mutants listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131865#pone.0131865.t002" target="_blank">Table 2</a>. “Yes” or “No” designates whether the ability to oxidize D-amino acid is concordant with the growth phenotypes of the specified <i>CgdaoΔ</i> mutant. The experiments were repeated twice and the representative data are shown.</p><p>^b CgDao1 showed the clearest concordant substrate specificity with <i>Cgdao1Δ</i> growth phenotype.</p><p>* Activity is at least two fold higher than the control activity of CgDao using L-amino acid as substrate.</p><p>Substrate specificity of Dao enzymes.</p

<i>DAO</i> genes are important for growth on D-amino acids.

<p>(A) Phenotype of R265 <i>daoΔ</i> mutants. (B) <i>CnDAO1</i> and <i>CnDAO3</i> 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 <i>DAO</i> 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.</p

Correlation between CORT levels during sensory phase and syrinx weight.

<p>Syrinx weight of black-capped chickadee in their first spring is correlated with CORT levels of their natal feathers (regression analysis, R² = 0.1, N = 20, p = 0.03).Circles represent birds from young forest; squares represent birds from mature forest.</p