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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

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.
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pone.0131865.g003: 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.

Mentions: In various fungi, the expression of DAO is induced in the growth media where a D-amino acid is the sole nitrogen source [38–40]. Since growth of R265 on D-Ala and D-Pro required CgDAO2, we examined the expression patterns of CgDAO2 in media containing D-Ala or D-Pro as the sole nitrogen source. The expression of CgDAO2 was detectable in the rich medium YPD, and its expression did not change substantially when the cells were shifted from YPD medium to defined medium containing ammonium sulfate as the sole nitrogen source for 6 h (Fig 3A). However, when R265 was shifted from YPD to D-Ala as the sole nitrogen source, expression of CgDAO2 increased at 3 h after transfer and maintained the increased levels for at least 25 hours. Similarly, expression of CgDAO2 increased slightly at 6 h after shifting from YPD to D-Pro and maintained the increased levels for at least for 25 hours. Therefore, CgDAO2 is required for the growth on D-Ala and D-Pro and its expression levels increase in the presence of these two D-amino acids.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131865.g003: 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.
Mentions: In various fungi, the expression of DAO is induced in the growth media where a D-amino acid is the sole nitrogen source [38–40]. Since growth of R265 on D-Ala and D-Pro required CgDAO2, we examined the expression patterns of CgDAO2 in media containing D-Ala or D-Pro as the sole nitrogen source. The expression of CgDAO2 was detectable in the rich medium YPD, and its expression did not change substantially when the cells were shifted from YPD medium to defined medium containing ammonium sulfate as the sole nitrogen source for 6 h (Fig 3A). However, when R265 was shifted from YPD to D-Ala as the sole nitrogen source, expression of CgDAO2 increased at 3 h after transfer and maintained the increased levels for at least 25 hours. Similarly, expression of CgDAO2 increased slightly at 6 h after shifting from YPD to D-Pro and maintained the increased levels for at least for 25 hours. Therefore, CgDAO2 is required for the growth on D-Ala and D-Pro and its expression levels increase in the presence of these two D-amino acids.

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