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Role of an expanded inositol transporter repertoire in Cryptococcus neoformans sexual reproduction and virulence.

Xue C, Liu T, Chen L, Li W, Liu I, Kronstad JW, Seyfang A, Heitman J - MBio (2010)

Bottom Line: Expression of ITR genes in a Saccharomyces cerevisiae itr1 itr2 mutant lacking inositol transport can complement the slow-growth phenotype of this strain, confirming that ITR genes are bona fide inositol transporters.Deletion of the inositol 1-phosphate synthase gene INO1 in an itr1 or itr1a mutant background compromised virulence in a murine inhalation model, indicating the importance of inositol sensing and acquisition for fungal infectivity.Our study provides a platform for further understanding the roles of inositol in fungal physiology and virulence.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA.

ABSTRACT
Cryptococcus neoformans and Cryptococcus gattii are globally distributed human fungal pathogens and the leading causes of fungal meningitis. Recent studies reveal that myo-inositol is an important factor for fungal sexual reproduction. That C. neoformans can utilize myo-inositol as a sole carbon source and the existence of abundant inositol in the human central nervous system suggest that inositol is important for Cryptococcus development and virulence. In accord with this central importance of inositol, an expanded myo-inositol transporter (ITR) gene family has been identified in Cryptococcus. This gene family contains two phylogenetically distinct groups, with a total of 10 or more members in C. neoformans and at least six members in the sibling species C. gattii. These inositol transporter genes are differentially expressed under inositol-inducing conditions based on quantitative real-time PCR analyses. Expression of ITR genes in a Saccharomyces cerevisiae itr1 itr2 mutant lacking inositol transport can complement the slow-growth phenotype of this strain, confirming that ITR genes are bona fide inositol transporters. Gene mutagenesis studies reveal that the Itr1 and Itr1A transporters are important for myo-inositol stimulation of mating and that functional redundancies among the myo-inositol transporters likely exist. Deletion of the inositol 1-phosphate synthase gene INO1 in an itr1 or itr1a mutant background compromised virulence in a murine inhalation model, indicating the importance of inositol sensing and acquisition for fungal infectivity. Our study provides a platform for further understanding the roles of inositol in fungal physiology and virulence.

No MeSH data available.


Related in: MedlinePlus

ITR genes from C. neoformans complement the growth defect of an S. cerevisiae itr1 itr2 mutant strain. S. cerevisiae itr1, itr1 itr2, and itr1 itr2 expressing vector pTH19 or one of the seven C. neoformans ITR genes were cultured in YPD or SD-Ura medium. Concentrations of overnight cultures were determined by measuring the optical density at 600 nm (OD600) and adjusted to the same cell density with YPD. Serial 10-fold dilutions were prepared, and 5 µl of each dilution was spotted on YPD plates and incubated at 30°C or 37°C for 48 h before photography. This assay was repeated multiple times, with similar results. Sc, S. cerevisiae; Cn, C. neoformans; wt, wild type.
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f5: ITR genes from C. neoformans complement the growth defect of an S. cerevisiae itr1 itr2 mutant strain. S. cerevisiae itr1, itr1 itr2, and itr1 itr2 expressing vector pTH19 or one of the seven C. neoformans ITR genes were cultured in YPD or SD-Ura medium. Concentrations of overnight cultures were determined by measuring the optical density at 600 nm (OD600) and adjusted to the same cell density with YPD. Serial 10-fold dilutions were prepared, and 5 µl of each dilution was spotted on YPD plates and incubated at 30°C or 37°C for 48 h before photography. This assay was repeated multiple times, with similar results. Sc, S. cerevisiae; Cn, C. neoformans; wt, wild type.

Mentions: To understand the potential role of the C. neoformans ITR gene family in inositol uptake, we generated S. cerevisiae itr1 itr2 double mutants lacking both inositol transporters. Such double mutants showed a growth defect in yeast extract-peptone-dextrose (YPD) at 37°C. Full-length cDNAs for all seven ITR genes from the C. neoformans H99 strain were cloned into the yeast expression vector pTH19 under the control of the ADH1 promoter and expressed in an S. cerevisiae itr1 itr2 double mutant strain. The heterologous expression of all seven ITR genes of H99 was confirmed by RT-PCR analyses (data not shown). Growth assays were performed for these S. cerevisiae strains expressing C. neoformans ITR genes, and the potential complementation of the S. cerevisiae itr1 itr2 growth defect phenotype was investigated. Expression of ITR1A and ITR3C fully complemented the growth defect of the S. cerevisiae itr1 itr2 mutant strain, while ITR3A and ITR3B partially rescued the slow growth on YPD medium (Fig. 5). Yeast strains expressing ITR1, ITR2, or ITR3, on the other hand, showed growth rates similar to that of the original S. cerevisiae itr1 itr2 mutant strain, suggesting no obvious complementation. These results suggest that Itr1A and Itr3C are important for inositol uptake, which is consistent with our qRT-PCR results suggesting that both ITR1A and ITR3C are highly expressed in response to the availability of environmental myo-inositol. The ITR genes that did not complement the growth defect of S. cerevisiae itr1 itr2 might be caused by either insufficient inositol uptake or expressed proteins that are unstable or not functional.


Role of an expanded inositol transporter repertoire in Cryptococcus neoformans sexual reproduction and virulence.

Xue C, Liu T, Chen L, Li W, Liu I, Kronstad JW, Seyfang A, Heitman J - MBio (2010)

ITR genes from C. neoformans complement the growth defect of an S. cerevisiae itr1 itr2 mutant strain. S. cerevisiae itr1, itr1 itr2, and itr1 itr2 expressing vector pTH19 or one of the seven C. neoformans ITR genes were cultured in YPD or SD-Ura medium. Concentrations of overnight cultures were determined by measuring the optical density at 600 nm (OD600) and adjusted to the same cell density with YPD. Serial 10-fold dilutions were prepared, and 5 µl of each dilution was spotted on YPD plates and incubated at 30°C or 37°C for 48 h before photography. This assay was repeated multiple times, with similar results. Sc, S. cerevisiae; Cn, C. neoformans; wt, wild type.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2912663&req=5

f5: ITR genes from C. neoformans complement the growth defect of an S. cerevisiae itr1 itr2 mutant strain. S. cerevisiae itr1, itr1 itr2, and itr1 itr2 expressing vector pTH19 or one of the seven C. neoformans ITR genes were cultured in YPD or SD-Ura medium. Concentrations of overnight cultures were determined by measuring the optical density at 600 nm (OD600) and adjusted to the same cell density with YPD. Serial 10-fold dilutions were prepared, and 5 µl of each dilution was spotted on YPD plates and incubated at 30°C or 37°C for 48 h before photography. This assay was repeated multiple times, with similar results. Sc, S. cerevisiae; Cn, C. neoformans; wt, wild type.
Mentions: To understand the potential role of the C. neoformans ITR gene family in inositol uptake, we generated S. cerevisiae itr1 itr2 double mutants lacking both inositol transporters. Such double mutants showed a growth defect in yeast extract-peptone-dextrose (YPD) at 37°C. Full-length cDNAs for all seven ITR genes from the C. neoformans H99 strain were cloned into the yeast expression vector pTH19 under the control of the ADH1 promoter and expressed in an S. cerevisiae itr1 itr2 double mutant strain. The heterologous expression of all seven ITR genes of H99 was confirmed by RT-PCR analyses (data not shown). Growth assays were performed for these S. cerevisiae strains expressing C. neoformans ITR genes, and the potential complementation of the S. cerevisiae itr1 itr2 growth defect phenotype was investigated. Expression of ITR1A and ITR3C fully complemented the growth defect of the S. cerevisiae itr1 itr2 mutant strain, while ITR3A and ITR3B partially rescued the slow growth on YPD medium (Fig. 5). Yeast strains expressing ITR1, ITR2, or ITR3, on the other hand, showed growth rates similar to that of the original S. cerevisiae itr1 itr2 mutant strain, suggesting no obvious complementation. These results suggest that Itr1A and Itr3C are important for inositol uptake, which is consistent with our qRT-PCR results suggesting that both ITR1A and ITR3C are highly expressed in response to the availability of environmental myo-inositol. The ITR genes that did not complement the growth defect of S. cerevisiae itr1 itr2 might be caused by either insufficient inositol uptake or expressed proteins that are unstable or not functional.

Bottom Line: Expression of ITR genes in a Saccharomyces cerevisiae itr1 itr2 mutant lacking inositol transport can complement the slow-growth phenotype of this strain, confirming that ITR genes are bona fide inositol transporters.Deletion of the inositol 1-phosphate synthase gene INO1 in an itr1 or itr1a mutant background compromised virulence in a murine inhalation model, indicating the importance of inositol sensing and acquisition for fungal infectivity.Our study provides a platform for further understanding the roles of inositol in fungal physiology and virulence.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina, USA.

ABSTRACT
Cryptococcus neoformans and Cryptococcus gattii are globally distributed human fungal pathogens and the leading causes of fungal meningitis. Recent studies reveal that myo-inositol is an important factor for fungal sexual reproduction. That C. neoformans can utilize myo-inositol as a sole carbon source and the existence of abundant inositol in the human central nervous system suggest that inositol is important for Cryptococcus development and virulence. In accord with this central importance of inositol, an expanded myo-inositol transporter (ITR) gene family has been identified in Cryptococcus. This gene family contains two phylogenetically distinct groups, with a total of 10 or more members in C. neoformans and at least six members in the sibling species C. gattii. These inositol transporter genes are differentially expressed under inositol-inducing conditions based on quantitative real-time PCR analyses. Expression of ITR genes in a Saccharomyces cerevisiae itr1 itr2 mutant lacking inositol transport can complement the slow-growth phenotype of this strain, confirming that ITR genes are bona fide inositol transporters. Gene mutagenesis studies reveal that the Itr1 and Itr1A transporters are important for myo-inositol stimulation of mating and that functional redundancies among the myo-inositol transporters likely exist. Deletion of the inositol 1-phosphate synthase gene INO1 in an itr1 or itr1a mutant background compromised virulence in a murine inhalation model, indicating the importance of inositol sensing and acquisition for fungal infectivity. Our study provides a platform for further understanding the roles of inositol in fungal physiology and virulence.

No MeSH data available.


Related in: MedlinePlus