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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

Itr1 and Itr1A promote mating in response to inositol. (A) Bilateral mating assays were performed in MS medium with all seven inositol transporter mutants and with double mutants lacking ino1 and each itr gene. Mating cultures were incubated at room temperature in the dark for 7 days before being examined by microscopy and photographed. Both mating filaments (40× magnification) and spores (insets, 400× magnification) were observed for all mating cultures. Mating of itr1, ino1 itr1, and ino1 itr1a mutants showed a defect in mating filament production and sporulation, suggesting an important role in inositol sensing. (B) Bilateral mating cultures of wild-type strains, ino1 itr1 mutants, and ino1 itr1a mutants were incubated under the same conditions as described in the legend to panel A for 14 days, and sporulation results were photographed under 200× magnification. White arrows indicate basidiospore chains; black arrows indicate basidia. (C) Cell fusion assays were performed for wild-type strains (YSB119 and YSB121) and itr1 mutants (CDX175 and CUX50). Cell fusion results were quantified from repeat assays, and representative images are shown.
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f6: Itr1 and Itr1A promote mating in response to inositol. (A) Bilateral mating assays were performed in MS medium with all seven inositol transporter mutants and with double mutants lacking ino1 and each itr gene. Mating cultures were incubated at room temperature in the dark for 7 days before being examined by microscopy and photographed. Both mating filaments (40× magnification) and spores (insets, 400× magnification) were observed for all mating cultures. Mating of itr1, ino1 itr1, and ino1 itr1a mutants showed a defect in mating filament production and sporulation, suggesting an important role in inositol sensing. (B) Bilateral mating cultures of wild-type strains, ino1 itr1 mutants, and ino1 itr1a mutants were incubated under the same conditions as described in the legend to panel A for 14 days, and sporulation results were photographed under 200× magnification. White arrows indicate basidiospore chains; black arrows indicate basidia. (C) Cell fusion assays were performed for wild-type strains (YSB119 and YSB121) and itr1 mutants (CDX175 and CUX50). Cell fusion results were quantified from repeat assays, and representative images are shown.

Mentions: Gene deletion mutations were generated by biolistic transformation and homologous recombination for all seven ITR genes in C. neoformans var. grubii in both the α and a mating-type backgrounds. Each individual mutation was not sufficient to block sexual reproduction based on mating assays with both MS and V8 mating media. Normal mating hyphae and spores were observed in all bilateral mutants by mutant mating assays. The exception was that bilateral mating between itr1 mutants exhibited reduced mating hypha production (Fig. 6A). Cell fusion assays showed that itr1 mutants have cell fusion efficiency similar to that of wild-type strains, suggesting that the mating defect could be caused by post-cell fusion events, such as insufficient dikaryotic hypha production or a meiosis defect (Fig. 6C). Because expression of ITR1 in an S. cerevisiae itr1 itr2 mutant strain failed to rescue the growth defect of that yeast strain, Itr1 may not be important for inositol uptake but may play other roles, such as functioning as an inositol sensor. The fact that ITR1 expression is repressed by high concentrations of inositol also suggests that Itr1 may be functional as an inositol sensor. To further understand the potential role of Itr1 in the regulation of other Itrs, qRT-PCR analysis was performed to test the expression of the other six ITR genes in the itr1 mutant background, both as single cultures and during mating, using the same approach as that used for wild-type strains (Fig. 4C and D). Our results revealed that ITR3, ITR3B, and ITR3C are expressed at a much higher level in itr1 single cultures than in the H99 wild-type strain. During mating, most ITR genes were highly expressed, with similar expression patterns in both itr1 mating cultures and wild-type strains, except that ITR3A was highly expressed only in itr1 mating cultures (Fig. 4). These results indicate that the ITR3 subgroup genes may be regulated by Itr1, which further suggests that Itr1 could be an inositol sensor that regulates other Itrs.


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)

Itr1 and Itr1A promote mating in response to inositol. (A) Bilateral mating assays were performed in MS medium with all seven inositol transporter mutants and with double mutants lacking ino1 and each itr gene. Mating cultures were incubated at room temperature in the dark for 7 days before being examined by microscopy and photographed. Both mating filaments (40× magnification) and spores (insets, 400× magnification) were observed for all mating cultures. Mating of itr1, ino1 itr1, and ino1 itr1a mutants showed a defect in mating filament production and sporulation, suggesting an important role in inositol sensing. (B) Bilateral mating cultures of wild-type strains, ino1 itr1 mutants, and ino1 itr1a mutants were incubated under the same conditions as described in the legend to panel A for 14 days, and sporulation results were photographed under 200× magnification. White arrows indicate basidiospore chains; black arrows indicate basidia. (C) Cell fusion assays were performed for wild-type strains (YSB119 and YSB121) and itr1 mutants (CDX175 and CUX50). Cell fusion results were quantified from repeat assays, and representative images are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f6: Itr1 and Itr1A promote mating in response to inositol. (A) Bilateral mating assays were performed in MS medium with all seven inositol transporter mutants and with double mutants lacking ino1 and each itr gene. Mating cultures were incubated at room temperature in the dark for 7 days before being examined by microscopy and photographed. Both mating filaments (40× magnification) and spores (insets, 400× magnification) were observed for all mating cultures. Mating of itr1, ino1 itr1, and ino1 itr1a mutants showed a defect in mating filament production and sporulation, suggesting an important role in inositol sensing. (B) Bilateral mating cultures of wild-type strains, ino1 itr1 mutants, and ino1 itr1a mutants were incubated under the same conditions as described in the legend to panel A for 14 days, and sporulation results were photographed under 200× magnification. White arrows indicate basidiospore chains; black arrows indicate basidia. (C) Cell fusion assays were performed for wild-type strains (YSB119 and YSB121) and itr1 mutants (CDX175 and CUX50). Cell fusion results were quantified from repeat assays, and representative images are shown.
Mentions: Gene deletion mutations were generated by biolistic transformation and homologous recombination for all seven ITR genes in C. neoformans var. grubii in both the α and a mating-type backgrounds. Each individual mutation was not sufficient to block sexual reproduction based on mating assays with both MS and V8 mating media. Normal mating hyphae and spores were observed in all bilateral mutants by mutant mating assays. The exception was that bilateral mating between itr1 mutants exhibited reduced mating hypha production (Fig. 6A). Cell fusion assays showed that itr1 mutants have cell fusion efficiency similar to that of wild-type strains, suggesting that the mating defect could be caused by post-cell fusion events, such as insufficient dikaryotic hypha production or a meiosis defect (Fig. 6C). Because expression of ITR1 in an S. cerevisiae itr1 itr2 mutant strain failed to rescue the growth defect of that yeast strain, Itr1 may not be important for inositol uptake but may play other roles, such as functioning as an inositol sensor. The fact that ITR1 expression is repressed by high concentrations of inositol also suggests that Itr1 may be functional as an inositol sensor. To further understand the potential role of Itr1 in the regulation of other Itrs, qRT-PCR analysis was performed to test the expression of the other six ITR genes in the itr1 mutant background, both as single cultures and during mating, using the same approach as that used for wild-type strains (Fig. 4C and D). Our results revealed that ITR3, ITR3B, and ITR3C are expressed at a much higher level in itr1 single cultures than in the H99 wild-type strain. During mating, most ITR genes were highly expressed, with similar expression patterns in both itr1 mating cultures and wild-type strains, except that ITR3A was highly expressed only in itr1 mating cultures (Fig. 4). These results indicate that the ITR3 subgroup genes may be regulated by Itr1, which further suggests that Itr1 could be an inositol sensor that regulates other Itrs.

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