Limits...
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 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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4489021&req=5

pone.0131865.g001: 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.

Mentions: Oxidative deamination of D-amino acids by Dao produces ammonia which can be used as the sole nitrogen source for growth. We deleted each of the three CgDAO genes in R265 and examined its effect on growth on all common D-amino acids. Deletion of individual CgDAO1, CgDAO2 or, CgDAO3 genes did not affect growth on any of the common L-amino acids (Table 2) indicating that DAO genes are not required for L-amino acids utilization. Deletion of CgDAO2 resulted in retarded growth on 11 different D-amino acids and reconstitution of CgDAO2 restored the growth on those D-amino acids (Fig 1A and Table 2) suggesting that CgDAO2 is the major DAO gene required for normal growth of R265 on those 11 D-amino acids. Deletion of CgDAO3 in R265 markedly reduced the growth on D-Glu and slightly affected the growth on D-D-Asp compared to the wild-type. Growth of Cgdao1Δ was also slightly affected in D-Asp. Therefore, D-Asp was the only D-amino acid with which all three single dao deletants showed a phenotype of reduced growth. Although deletion of each CgDAO gene separately did not clearly affect the growth of R265 on D-Asn, D-Trp or D-Tyr, the triple CgDAO gene deletion resulted in severe growth retardation on those three D-amino acids. Interestingly, all R265 dao single deletants grew well on D-Arg, D-Gln, D-Ile and D-Lys (Table 2). These amino acids, except for D-Ile, all contain a side-chain amine group that could be used as a nitrogen source. These observations indicate that DAO genes are important for growth of R265 on all common D-amino acids that lack the side-chain amine group except for D-Ile. It is possible that additional unidentified D-amino acid oxidases or other enzyme(s) in R265 may convert D-Ile to a useable nitrogen source.


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131865.g001: 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.
Mentions: Oxidative deamination of D-amino acids by Dao produces ammonia which can be used as the sole nitrogen source for growth. We deleted each of the three CgDAO genes in R265 and examined its effect on growth on all common D-amino acids. Deletion of individual CgDAO1, CgDAO2 or, CgDAO3 genes did not affect growth on any of the common L-amino acids (Table 2) indicating that DAO genes are not required for L-amino acids utilization. Deletion of CgDAO2 resulted in retarded growth on 11 different D-amino acids and reconstitution of CgDAO2 restored the growth on those D-amino acids (Fig 1A and Table 2) suggesting that CgDAO2 is the major DAO gene required for normal growth of R265 on those 11 D-amino acids. Deletion of CgDAO3 in R265 markedly reduced the growth on D-Glu and slightly affected the growth on D-D-Asp compared to the wild-type. Growth of Cgdao1Δ was also slightly affected in D-Asp. Therefore, D-Asp was the only D-amino acid with which all three single dao deletants showed a phenotype of reduced growth. Although deletion of each CgDAO gene separately did not clearly affect the growth of R265 on D-Asn, D-Trp or D-Tyr, the triple CgDAO gene deletion resulted in severe growth retardation on those three D-amino acids. Interestingly, all R265 dao single deletants grew well on D-Arg, D-Gln, D-Ile and D-Lys (Table 2). These amino acids, except for D-Ile, all contain a side-chain amine group that could be used as a nitrogen source. These observations indicate that DAO genes are important for growth of R265 on all common D-amino acids that lack the side-chain amine group except for D-Ile. It is possible that additional unidentified D-amino acid oxidases or other enzyme(s) in R265 may convert D-Ile to a useable nitrogen source.

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