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Carbon dioxide is a powerful inducer of monokaryotic hyphae and spore development in Cryptococcus gattii and carbonic anhydrase activity is dispensable in this dimorphic transition.

Ren P, Chaturvedi V, Chaturvedi S - PLoS ONE (2014)

Bottom Line: Both CAN1 and CAN2 were dispensable for CO2 induced morphogenetic transitions.However, C. gattii CAN2 was essential for growth in ambient air similar to its reported role in C. neoformans.Both can1 and can2 mutants retained full pathogenic potential in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Mycology Laboratory, Wadsworth Center, New York State Department of Health, 120 New Scotland Avenue, Albany, New York, United States of America.

ABSTRACT
Cryptococcus gattii is unique among human pathogenic fungi with specialized ecological niche on trees. Since leaves concentrate CO2, we investigated the role of this gaseous molecule in C. gattii biology and virulence. We focused on the genetic analyses of β-carbonic anhydrase (β-CA) encoded by C. gattii CAN1 and CAN2 as later is critical for CO2 sensing in a closely related pathogen C. neoformans. High CO2 conditions induced robust development of monokaryotic hyphae and spores in C. gattii. Conversely, high CO2 completely repressed hyphae development in sexual mating. Both CAN1 and CAN2 were dispensable for CO2 induced morphogenetic transitions. However, C. gattii CAN2 was essential for growth in ambient air similar to its reported role in C. neoformans. Both can1 and can2 mutants retained full pathogenic potential in vitro and in vivo. These results provide insight into C. gattii adaptation for arboreal growth and production of infectious propagules by β-CA independent mechanism(s).

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CAN2 is critical for fatty acid biosynthesis but not essential for adenyl cycalse (CAC1) gene expression.(a). Palmitic acid barely restored can2 mutant growth in ambient air.C. gattii strains grown overnight in high CO2 were collected, washed, serially diluted, and spotted on YPD medium containing palmitate with 1% Tween 80 as surfactant. Cells were incubated at 30°C in ambient air for 5 days. The growth defect of can2 mutant was barely rescued in the presence of low but not high concentration of palmitate in ambient air. The halo surrounding the growth patches of WT and reconstituted strains reflects efficient utilization of fatty acids from media. (b)Semi-quantitative RT-PCR confirmed CAC1 gene expression is independent of CAN2. C. gattii WT, can2 mutant, and can2+CAN2 reconstituted strain were grown in YPD broth in high CO2 or in YPD broth containing 1 mM sodium palmitate in ambient air, for 3 days at 30°C. Total RNA was isolated and reverse transcribed (cDNA) with 100-ng aliquots in 1∶10 serial dilutions. SOD1 was used as a loading control. (c)Semi-quantitative RT-PCR confirmed CAN2 gene expression is not regulated by CO2. CAN2 transcript in total RNA was determined from C. gattii WT strain grown in various conditions as indicated. SOD1 was used as a loading control.
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pone-0113147-g002: CAN2 is critical for fatty acid biosynthesis but not essential for adenyl cycalse (CAC1) gene expression.(a). Palmitic acid barely restored can2 mutant growth in ambient air.C. gattii strains grown overnight in high CO2 were collected, washed, serially diluted, and spotted on YPD medium containing palmitate with 1% Tween 80 as surfactant. Cells were incubated at 30°C in ambient air for 5 days. The growth defect of can2 mutant was barely rescued in the presence of low but not high concentration of palmitate in ambient air. The halo surrounding the growth patches of WT and reconstituted strains reflects efficient utilization of fatty acids from media. (b)Semi-quantitative RT-PCR confirmed CAC1 gene expression is independent of CAN2. C. gattii WT, can2 mutant, and can2+CAN2 reconstituted strain were grown in YPD broth in high CO2 or in YPD broth containing 1 mM sodium palmitate in ambient air, for 3 days at 30°C. Total RNA was isolated and reverse transcribed (cDNA) with 100-ng aliquots in 1∶10 serial dilutions. SOD1 was used as a loading control. (c)Semi-quantitative RT-PCR confirmed CAN2 gene expression is not regulated by CO2. CAN2 transcript in total RNA was determined from C. gattii WT strain grown in various conditions as indicated. SOD1 was used as a loading control.

Mentions: We reasoned that the inability of the can2 mutant to grow in air could be due to limiting amounts of bicarbonate, a critical substrate required for the synthesis of several cellular carboxylases important in metabolism [31]. Bicarbonate is also a critical substrate for CAC1 gene activation, and that in turn leads to the synthesis of cAMP, a ubiquitous second messenger that regulates a large variety of essential physiological processes [21], [22]. Interestingly, addition of exogenous cAMP (2–10 mM) or sodium bicarbonate (1–10 mM) either singly or in combination, failed to complement the growth defect of the can2 mutant in ambient air. Similarly, addition of various cellular metabolites and carbon sources, including citrate, succinate, oxalaacetate, malate, α-ketoglutarate failed to complement the growth defect of the can2 mutant (data not shown). In contrast to the report published for C. neoformans, the growth defect of the can2 mutant was barely rescued by addition of exogenous fatty acids, 0.1 mM and 1 mM palmitate (Fig. 2a), indicating that CAN2 is essential for fatty-acid biosynthetic processes in ambient air in C. gattii. We observed a clear zone surrounding the colonies of C. gattii WT and can2+CAN2 reconstituted strains (2 mM and 5 mM palmitate) (Fig. 2a). This might be due to the fact that WT and reconstitute strains were able to utilize fatty acids from media resulting in clear zone surrounding the growth.


Carbon dioxide is a powerful inducer of monokaryotic hyphae and spore development in Cryptococcus gattii and carbonic anhydrase activity is dispensable in this dimorphic transition.

Ren P, Chaturvedi V, Chaturvedi S - PLoS ONE (2014)

CAN2 is critical for fatty acid biosynthesis but not essential for adenyl cycalse (CAC1) gene expression.(a). Palmitic acid barely restored can2 mutant growth in ambient air.C. gattii strains grown overnight in high CO2 were collected, washed, serially diluted, and spotted on YPD medium containing palmitate with 1% Tween 80 as surfactant. Cells were incubated at 30°C in ambient air for 5 days. The growth defect of can2 mutant was barely rescued in the presence of low but not high concentration of palmitate in ambient air. The halo surrounding the growth patches of WT and reconstituted strains reflects efficient utilization of fatty acids from media. (b)Semi-quantitative RT-PCR confirmed CAC1 gene expression is independent of CAN2. C. gattii WT, can2 mutant, and can2+CAN2 reconstituted strain were grown in YPD broth in high CO2 or in YPD broth containing 1 mM sodium palmitate in ambient air, for 3 days at 30°C. Total RNA was isolated and reverse transcribed (cDNA) with 100-ng aliquots in 1∶10 serial dilutions. SOD1 was used as a loading control. (c)Semi-quantitative RT-PCR confirmed CAN2 gene expression is not regulated by CO2. CAN2 transcript in total RNA was determined from C. gattii WT strain grown in various conditions as indicated. SOD1 was used as a loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4257545&req=5

pone-0113147-g002: CAN2 is critical for fatty acid biosynthesis but not essential for adenyl cycalse (CAC1) gene expression.(a). Palmitic acid barely restored can2 mutant growth in ambient air.C. gattii strains grown overnight in high CO2 were collected, washed, serially diluted, and spotted on YPD medium containing palmitate with 1% Tween 80 as surfactant. Cells were incubated at 30°C in ambient air for 5 days. The growth defect of can2 mutant was barely rescued in the presence of low but not high concentration of palmitate in ambient air. The halo surrounding the growth patches of WT and reconstituted strains reflects efficient utilization of fatty acids from media. (b)Semi-quantitative RT-PCR confirmed CAC1 gene expression is independent of CAN2. C. gattii WT, can2 mutant, and can2+CAN2 reconstituted strain were grown in YPD broth in high CO2 or in YPD broth containing 1 mM sodium palmitate in ambient air, for 3 days at 30°C. Total RNA was isolated and reverse transcribed (cDNA) with 100-ng aliquots in 1∶10 serial dilutions. SOD1 was used as a loading control. (c)Semi-quantitative RT-PCR confirmed CAN2 gene expression is not regulated by CO2. CAN2 transcript in total RNA was determined from C. gattii WT strain grown in various conditions as indicated. SOD1 was used as a loading control.
Mentions: We reasoned that the inability of the can2 mutant to grow in air could be due to limiting amounts of bicarbonate, a critical substrate required for the synthesis of several cellular carboxylases important in metabolism [31]. Bicarbonate is also a critical substrate for CAC1 gene activation, and that in turn leads to the synthesis of cAMP, a ubiquitous second messenger that regulates a large variety of essential physiological processes [21], [22]. Interestingly, addition of exogenous cAMP (2–10 mM) or sodium bicarbonate (1–10 mM) either singly or in combination, failed to complement the growth defect of the can2 mutant in ambient air. Similarly, addition of various cellular metabolites and carbon sources, including citrate, succinate, oxalaacetate, malate, α-ketoglutarate failed to complement the growth defect of the can2 mutant (data not shown). In contrast to the report published for C. neoformans, the growth defect of the can2 mutant was barely rescued by addition of exogenous fatty acids, 0.1 mM and 1 mM palmitate (Fig. 2a), indicating that CAN2 is essential for fatty-acid biosynthetic processes in ambient air in C. gattii. We observed a clear zone surrounding the colonies of C. gattii WT and can2+CAN2 reconstituted strains (2 mM and 5 mM palmitate) (Fig. 2a). This might be due to the fact that WT and reconstitute strains were able to utilize fatty acids from media resulting in clear zone surrounding the growth.

Bottom Line: Both CAN1 and CAN2 were dispensable for CO2 induced morphogenetic transitions.However, C. gattii CAN2 was essential for growth in ambient air similar to its reported role in C. neoformans.Both can1 and can2 mutants retained full pathogenic potential in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Mycology Laboratory, Wadsworth Center, New York State Department of Health, 120 New Scotland Avenue, Albany, New York, United States of America.

ABSTRACT
Cryptococcus gattii is unique among human pathogenic fungi with specialized ecological niche on trees. Since leaves concentrate CO2, we investigated the role of this gaseous molecule in C. gattii biology and virulence. We focused on the genetic analyses of β-carbonic anhydrase (β-CA) encoded by C. gattii CAN1 and CAN2 as later is critical for CO2 sensing in a closely related pathogen C. neoformans. High CO2 conditions induced robust development of monokaryotic hyphae and spores in C. gattii. Conversely, high CO2 completely repressed hyphae development in sexual mating. Both CAN1 and CAN2 were dispensable for CO2 induced morphogenetic transitions. However, C. gattii CAN2 was essential for growth in ambient air similar to its reported role in C. neoformans. Both can1 and can2 mutants retained full pathogenic potential in vitro and in vivo. These results provide insight into C. gattii adaptation for arboreal growth and production of infectious propagules by β-CA independent mechanism(s).

Show MeSH
Related in: MedlinePlus