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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

Virulence of the triple dao deletant of R265 is reduced.(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: Cgdao1ΔCgdao2ΔCgdao3Δ triple deletant; C1645: Cgdao1Cgdao3Δ double deletant (derived from C1624 by reconstituting CgDAO2). The experiments were repeated twice and the representative data are shown.
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pone.0131865.g006: Virulence of the triple dao deletant of R265 is reduced.(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: Cgdao1ΔCgdao2ΔCgdao3Δ triple deletant; C1645: Cgdao1Cgdao3Δ double deletant (derived from C1624 by reconstituting CgDAO2). The experiments were repeated twice and the representative data are shown.

Mentions: Since CgDAO2 was the major DAO gene in R265, we examined the role of CgDAO2 in virulence using murine models. Deletion of CgDAO2 did not affect virulence in mice infected either by intrapharyngeal aspiration (IPh) or intravenous injection (IV) (Fig 6A). Similarly, neither the deletion of CgDAO1 nor CgDAO3 affected virulence of R265 (data now shown). Interestingly, virulence of the Cgdao1ΔCgdao2ΔCgdao3Δ triple deletant (C1624) was reduced compared to the wild-type in both IPh (p = 0.018, log-rank test) and IV (p = 0.041, log-rank test) models while complementation of CgDAO2 in the triple deletant (C1645) restored the virulence (Fig 6B). This suggests that although the individual DAO gene is not required for the virulence of R265, the three CgDAO genes cooperatively contribute to virulence. On the other hand, the Cndao1ΔCndao3Δ double deletant or the Cndao1ΔCndao2ΔCndao3Δ triple deletant exhibited virulence similar to H99 suggesting that DAO genes in H99 do not play a role in pathogenicity (S4A Fig).


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)

Virulence of the triple dao deletant of R265 is reduced.(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: Cgdao1ΔCgdao2ΔCgdao3Δ triple deletant; C1645: Cgdao1Cgdao3Δ double deletant (derived from C1624 by reconstituting CgDAO2). The experiments were repeated twice and the representative data are shown.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0131865.g006: Virulence of the triple dao deletant of R265 is reduced.(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: Cgdao1ΔCgdao2ΔCgdao3Δ triple deletant; C1645: Cgdao1Cgdao3Δ double deletant (derived from C1624 by reconstituting CgDAO2). The experiments were repeated twice and the representative data are shown.
Mentions: Since CgDAO2 was the major DAO gene in R265, we examined the role of CgDAO2 in virulence using murine models. Deletion of CgDAO2 did not affect virulence in mice infected either by intrapharyngeal aspiration (IPh) or intravenous injection (IV) (Fig 6A). Similarly, neither the deletion of CgDAO1 nor CgDAO3 affected virulence of R265 (data now shown). Interestingly, virulence of the Cgdao1ΔCgdao2ΔCgdao3Δ triple deletant (C1624) was reduced compared to the wild-type in both IPh (p = 0.018, log-rank test) and IV (p = 0.041, log-rank test) models while complementation of CgDAO2 in the triple deletant (C1645) restored the virulence (Fig 6B). This suggests that although the individual DAO gene is not required for the virulence of R265, the three CgDAO genes cooperatively contribute to virulence. On the other hand, the Cndao1ΔCndao3Δ double deletant or the Cndao1ΔCndao2ΔCndao3Δ triple deletant exhibited virulence similar to H99 suggesting that DAO genes in H99 do not play a role in pathogenicity (S4A Fig).

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