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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

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.
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pone.0131865.g005: 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.

Mentions: Since CgDAO2 is the major DAO gene in R265 and overexpression of CnDAO2 complemented the Cgdao2Δ phenotype, we further explored the function of DAO2 genes in both species. Since overexpression of CgDAO2 using the GPD promoter enabled the H99 strain to utilize several D-amino acids, we tested if using the native CgDAO2 promoter could have a similar effect. Insertion of the entire CgDAO2 gene into the Cndao2Δ locus in H99 (Fig 5A upper panel; R2(p)-R2; strain C1720) enabled its growth on D-Ala (Fig 5B). This result indicates that a single copy of the CgDAO2 gene enables H99 to utilize D-Ala. When the CgDAO2 promoter in C1720 was swapped with that of CnDAO2, the resulting strain, C1728 (H2(p)-R2), grew better on D-Ala than H99 but weaker than C1720. The transcriptional levels of CgDAO2 determined by quantitative RT-PCR were higher in C1720 than in C1728 when the cells were grown in ammonium sulfate (Fig 5C). The transcription levels of CgDAO2 were slightly induced in C1720 by D-Ala but not in C1728. These data suggest that the apparent strength of the CgDAO2 promoter relative to that of CnDAO2 may explain the growth difference between C1720 and C1728 on 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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131865.g005: 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.
Mentions: Since CgDAO2 is the major DAO gene in R265 and overexpression of CnDAO2 complemented the Cgdao2Δ phenotype, we further explored the function of DAO2 genes in both species. Since overexpression of CgDAO2 using the GPD promoter enabled the H99 strain to utilize several D-amino acids, we tested if using the native CgDAO2 promoter could have a similar effect. Insertion of the entire CgDAO2 gene into the Cndao2Δ locus in H99 (Fig 5A upper panel; R2(p)-R2; strain C1720) enabled its growth on D-Ala (Fig 5B). This result indicates that a single copy of the CgDAO2 gene enables H99 to utilize D-Ala. When the CgDAO2 promoter in C1720 was swapped with that of CnDAO2, the resulting strain, C1728 (H2(p)-R2), grew better on D-Ala than H99 but weaker than C1720. The transcriptional levels of CgDAO2 determined by quantitative RT-PCR were higher in C1720 than in C1728 when the cells were grown in ammonium sulfate (Fig 5C). The transcription levels of CgDAO2 were slightly induced in C1720 by D-Ala but not in C1728. These data suggest that the apparent strength of the CgDAO2 promoter relative to that of CnDAO2 may explain the growth difference between C1720 and C1728 on 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