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

Chang YC, Khanal Lamichhane A, Bradley J, Rodgers L, Ngamskulrungroj P, Kwon-Chung KJ - PLoS ONE (2015)

Bottom Line: Replacing the H99 DAO2 gene with a single copy of the R265 DAO2 gene also enabled its utilization of several D-amino acids.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Microbiology Section, Laboratory of Clinical Infectious Diseases, NIAID, NIH, Bethesda, MD, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0131865.g002: 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.

Mentions: D-amino acids have been reported to be toxic to many microorganisms including fungi and bacteria [34–37]. We evaluated the toxicity of six representative amino acids including D-Pro and D-Ala which poorly supported the growth of Cgdao2Δ, D-Trp which affected growth only on the triple deletant, as well as D-Asn, D-Asp and D-Glu, which failed to support growth of Cndao1Δ or Cndao3Δ mutant (Fig 1). The strains were spotted on defined agar media containing 2% glucose and 10 mM ammonium sulfate supplemented with or without 100 mM of the selected D- or L-amino acid. Growth of H99 was not affected by the presence of six D-amino acids tested or their L-enantiomers (Fig 2 and S2 Fig). In contrast, addition of D-Glu or D-Ala slightly reduced the growth of wild-type R265. Furthermore, growth of Cndao3Δ was slightly reduced in the presence of 100 mM D-Glu whereas growth of Cgdao2Δ on D-Ala and Cgdao3Δ on D-Glu was slower compared to the R265 strain (Fig 2). Triple and single deletants of DAO behaved similarly suggesting no additive effects from the deletion of more than one CgDAO gene. Therefore, both CnDAO3 and CgDAO3 influenced growth and detoxification of D-Glu in H99 and R265 respectively. In contrast, only CgDAO2 but not CnDAO2 influenced growth and detoxification of D-Ala.


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

Chang YC, Khanal Lamichhane A, Bradley J, Rodgers L, Ngamskulrungroj P, Kwon-Chung KJ - PLoS ONE (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4489021&req=5

pone.0131865.g002: 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.
Mentions: D-amino acids have been reported to be toxic to many microorganisms including fungi and bacteria [34–37]. We evaluated the toxicity of six representative amino acids including D-Pro and D-Ala which poorly supported the growth of Cgdao2Δ, D-Trp which affected growth only on the triple deletant, as well as D-Asn, D-Asp and D-Glu, which failed to support growth of Cndao1Δ or Cndao3Δ mutant (Fig 1). The strains were spotted on defined agar media containing 2% glucose and 10 mM ammonium sulfate supplemented with or without 100 mM of the selected D- or L-amino acid. Growth of H99 was not affected by the presence of six D-amino acids tested or their L-enantiomers (Fig 2 and S2 Fig). In contrast, addition of D-Glu or D-Ala slightly reduced the growth of wild-type R265. Furthermore, growth of Cndao3Δ was slightly reduced in the presence of 100 mM D-Glu whereas growth of Cgdao2Δ on D-Ala and Cgdao3Δ on D-Glu was slower compared to the R265 strain (Fig 2). Triple and single deletants of DAO behaved similarly suggesting no additive effects from the deletion of more than one CgDAO gene. Therefore, both CnDAO3 and CgDAO3 influenced growth and detoxification of D-Glu in H99 and R265 respectively. In contrast, only CgDAO2 but not CnDAO2 influenced growth and detoxification of D-Ala.

Bottom Line: Replacing the H99 DAO2 gene with a single copy of the R265 DAO2 gene also enabled its utilization of several D-amino acids.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Microbiology Section, Laboratory of Clinical Infectious Diseases, NIAID, NIH, Bethesda, MD, United States of America.

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.

No MeSH data available.


Related in: MedlinePlus