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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 mutant strains are still virulent in mice. (A and B) Female A/JCr mice were inoculated intranasally with the following strains: H99, itr1 mutant (CUX7), itr1a mutant (CDX99), itr2 mutant (CDX103), itr3 mutant (CDX105), itr3a mutant (CDX102), itr3b mutant (CDX101), itr3c mutant (CUX42), ino1 mutant (UBCINO1), ino1 itr1 double mutant (CUX17), and ino1 itr1a double mutant (CUX23). Two experiments were performed to finish the virulence test for all above-mentioned strains. Groups of 10 mice were infected with each strain. Animals were monitored for clinical signs of cryptococcal infection and sacrificed at predetermined clinical end points that predict imminent mortality. (C) Fungal burdens in organs infected by ino1 itr1 and ino1 itr1a mutants were compared with those in organs infected by H99 at the end point of the infection. The numbers of colony-forming units (CFU) per gram of organ tissue were measured in brain, lung, and spleen homogenates. Each error bar indicates the standard error of the mean for values obtained from testing three animals.
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f7: itr mutant strains are still virulent in mice. (A and B) Female A/JCr mice were inoculated intranasally with the following strains: H99, itr1 mutant (CUX7), itr1a mutant (CDX99), itr2 mutant (CDX103), itr3 mutant (CDX105), itr3a mutant (CDX102), itr3b mutant (CDX101), itr3c mutant (CUX42), ino1 mutant (UBCINO1), ino1 itr1 double mutant (CUX17), and ino1 itr1a double mutant (CUX23). Two experiments were performed to finish the virulence test for all above-mentioned strains. Groups of 10 mice were infected with each strain. Animals were monitored for clinical signs of cryptococcal infection and sacrificed at predetermined clinical end points that predict imminent mortality. (C) Fungal burdens in organs infected by ino1 itr1 and ino1 itr1a mutants were compared with those in organs infected by H99 at the end point of the infection. The numbers of colony-forming units (CFU) per gram of organ tissue were measured in brain, lung, and spleen homogenates. Each error bar indicates the standard error of the mean for values obtained from testing three animals.

Mentions: Although none of the itr mutants generated showed significant effects on several key virulence factors, such as capsule and melanin production and growth at 37°C (data not shown), their potential involvement in fungal virulence was assessed because virulence is a complex trait. All seven itr mutants (itr1, itr1a, itr2, itr3, itr3a, itr3b, and itr3c) were examined in a murine inhalation model of systemic C. neoformans infection. Groups of 10 female A/JCr mice were intranasally inoculated with 105 yeast cells from each strain, and animals were monitored daily. As previously demonstrated, all mice infected with the wild-type strain H99α survived between 16 and 22 days (39, 40). Mice infected with the mutants exhibited mortality rates similar to those of mice infected with the wild-type H99 strain, indicating that none of these seven itr mutants are attenuated for virulence of C. neoformans under these conditions (Fig. 7). It is possible that the functional redundancy of this gene family or the existence of the inositol biosynthesis pathway is sufficient for the virulence of each single mutant.


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 mutant strains are still virulent in mice. (A and B) Female A/JCr mice were inoculated intranasally with the following strains: H99, itr1 mutant (CUX7), itr1a mutant (CDX99), itr2 mutant (CDX103), itr3 mutant (CDX105), itr3a mutant (CDX102), itr3b mutant (CDX101), itr3c mutant (CUX42), ino1 mutant (UBCINO1), ino1 itr1 double mutant (CUX17), and ino1 itr1a double mutant (CUX23). Two experiments were performed to finish the virulence test for all above-mentioned strains. Groups of 10 mice were infected with each strain. Animals were monitored for clinical signs of cryptococcal infection and sacrificed at predetermined clinical end points that predict imminent mortality. (C) Fungal burdens in organs infected by ino1 itr1 and ino1 itr1a mutants were compared with those in organs infected by H99 at the end point of the infection. The numbers of colony-forming units (CFU) per gram of organ tissue were measured in brain, lung, and spleen homogenates. Each error bar indicates the standard error of the mean for values obtained from testing three animals.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: itr mutant strains are still virulent in mice. (A and B) Female A/JCr mice were inoculated intranasally with the following strains: H99, itr1 mutant (CUX7), itr1a mutant (CDX99), itr2 mutant (CDX103), itr3 mutant (CDX105), itr3a mutant (CDX102), itr3b mutant (CDX101), itr3c mutant (CUX42), ino1 mutant (UBCINO1), ino1 itr1 double mutant (CUX17), and ino1 itr1a double mutant (CUX23). Two experiments were performed to finish the virulence test for all above-mentioned strains. Groups of 10 mice were infected with each strain. Animals were monitored for clinical signs of cryptococcal infection and sacrificed at predetermined clinical end points that predict imminent mortality. (C) Fungal burdens in organs infected by ino1 itr1 and ino1 itr1a mutants were compared with those in organs infected by H99 at the end point of the infection. The numbers of colony-forming units (CFU) per gram of organ tissue were measured in brain, lung, and spleen homogenates. Each error bar indicates the standard error of the mean for values obtained from testing three animals.
Mentions: Although none of the itr mutants generated showed significant effects on several key virulence factors, such as capsule and melanin production and growth at 37°C (data not shown), their potential involvement in fungal virulence was assessed because virulence is a complex trait. All seven itr mutants (itr1, itr1a, itr2, itr3, itr3a, itr3b, and itr3c) were examined in a murine inhalation model of systemic C. neoformans infection. Groups of 10 female A/JCr mice were intranasally inoculated with 105 yeast cells from each strain, and animals were monitored daily. As previously demonstrated, all mice infected with the wild-type strain H99α survived between 16 and 22 days (39, 40). Mice infected with the mutants exhibited mortality rates similar to those of mice infected with the wild-type H99 strain, indicating that none of these seven itr mutants are attenuated for virulence of C. neoformans under these conditions (Fig. 7). It is possible that the functional redundancy of this gene family or the existence of the inositol biosynthesis pathway is sufficient for the virulence of each single mutant.

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