Limits...
Cell cycle-independent phospho-regulation of Fkh2 during hyphal growth regulates Candida albicans pathogenesis.

Greig JA, Sudbery IM, Richardson JP, Naglik JR, Wang Y, Sudbery PE - PLoS Pathog. (2015)

Bottom Line: We confirmed that these changes in gene expression resulted in corresponding defects in pathogenic processes.Furthermore, we identified that Fkh2 interacts with the chromatin modifier Pob3 in a phosphorylation-dependent manner, thereby providing a possible mechanism by which the phosphorylation of Fkh2 regulates its specificity.Thus, we have discovered a novel cell cycle-independent phospho-regulatory event that subverts a key component of the cell cycle machinery to a role in the switch from commensalism to pathogenicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, United Kingdom; Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore.

ABSTRACT
The opportunistic human fungal pathogen, Candida albicans, undergoes morphological and transcriptional adaptation in the switch from commensalism to pathogenicity. Although previous gene-knockout studies have identified many factors involved in this transformation, it remains unclear how these factors are regulated to coordinate the switch. Investigating morphogenetic control by post-translational phosphorylation has generated important regulatory insights into this process, especially focusing on coordinated control by the cyclin-dependent kinase Cdc28. Here we have identified the Fkh2 transcription factor as a regulatory target of both Cdc28 and the cell wall biosynthesis kinase Cbk1, in a role distinct from its conserved function in cell cycle progression. In stationary phase yeast cells 2D gel electrophoresis shows that there is a diverse pool of Fkh2 phospho-isoforms. For a short window on hyphal induction, far before START in the cell cycle, the phosphorylation profile is transformed before reverting to the yeast profile. This transformation does not occur when stationary phase cells are reinoculated into fresh medium supporting yeast growth. Mass spectrometry and mutational analyses identified residues phosphorylated by Cdc28 and Cbk1. Substitution of these residues with non-phosphorylatable alanine altered the yeast phosphorylation profile and abrogated the characteristic transformation to the hyphal profile. Transcript profiling of the phosphorylation site mutant revealed that the hyphal phosphorylation profile is required for the expression of genes involved in pathogenesis, host interaction and biofilm formation. We confirmed that these changes in gene expression resulted in corresponding defects in pathogenic processes. Furthermore, we identified that Fkh2 interacts with the chromatin modifier Pob3 in a phosphorylation-dependent manner, thereby providing a possible mechanism by which the phosphorylation of Fkh2 regulates its specificity. Thus, we have discovered a novel cell cycle-independent phospho-regulatory event that subverts a key component of the cell cycle machinery to a role in the switch from commensalism to pathogenicity.

Show MeSH

Related in: MedlinePlus

The effect of inhibiting Cdc28 or the removal of Cdc28 cyclins on Fkh2 phosphorylation.Fkh2 from cells of the indicated genotype and culture condition was fractionated by 2D gels. The cdc28-as1 strain was treated with 30 µM 1NM-PP1 to inhibit Cdc28. Note it is not possible to grow cells to stationary phase with Cdc28 inhibited. The CLN3-sd strain was grown to stationary phase overnight in YEPD which allows partial de-repression of the MET3 promoter regulating CLN3 expression [76]. For hyphal and yeast growth cells were inoculated from these stationary phase cultures into YEPD medium containing 2.5 mM methionine and 0.5 mM cysteine to repress CLN3 expression.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4305328&req=5

ppat.1004630.g003: The effect of inhibiting Cdc28 or the removal of Cdc28 cyclins on Fkh2 phosphorylation.Fkh2 from cells of the indicated genotype and culture condition was fractionated by 2D gels. The cdc28-as1 strain was treated with 30 µM 1NM-PP1 to inhibit Cdc28. Note it is not possible to grow cells to stationary phase with Cdc28 inhibited. The CLN3-sd strain was grown to stationary phase overnight in YEPD which allows partial de-repression of the MET3 promoter regulating CLN3 expression [76]. For hyphal and yeast growth cells were inoculated from these stationary phase cultures into YEPD medium containing 2.5 mM methionine and 0.5 mM cysteine to repress CLN3 expression.

Mentions: We next examined the Fkh2 2D profile in the cdc28-as1 mutant, and in mutants in which one of the Cdc28 G1 cyclins was either deleted (hgc1ΔΔ and ccn1ΔΔ) or down regulated using the MET3 promoter (CLN3-sd) (Fig. 3). The results showed that both inhibition of Cdc28 or lack of either Ccn1 or Cln3 prevented the shift to the hyphal profile, but there was less of an effect on the early hyphal profile in cells that lack Hgc1. Thus, the data support the conclusion that Cdc28 acts on Fkh2 early after hyphal induction, but suggest both Cln3 and Ccn1 are required to partner Cdc28. Inhibition of Cdc28 or lack of Cln3 also had a major effect on the Fkh2 profile in growing yeast cells, similar to the effect of the Fkh2(6A) mutant in these cells. Thus, while Cdc28 mediated phosphorylation is necessary for the transition to the early hyphal pattern, Fkh2 is also phosphorylated by Cdc28 during yeast growth, as would be expected from previous studies in S. cerevisiae.


Cell cycle-independent phospho-regulation of Fkh2 during hyphal growth regulates Candida albicans pathogenesis.

Greig JA, Sudbery IM, Richardson JP, Naglik JR, Wang Y, Sudbery PE - PLoS Pathog. (2015)

The effect of inhibiting Cdc28 or the removal of Cdc28 cyclins on Fkh2 phosphorylation.Fkh2 from cells of the indicated genotype and culture condition was fractionated by 2D gels. The cdc28-as1 strain was treated with 30 µM 1NM-PP1 to inhibit Cdc28. Note it is not possible to grow cells to stationary phase with Cdc28 inhibited. The CLN3-sd strain was grown to stationary phase overnight in YEPD which allows partial de-repression of the MET3 promoter regulating CLN3 expression [76]. For hyphal and yeast growth cells were inoculated from these stationary phase cultures into YEPD medium containing 2.5 mM methionine and 0.5 mM cysteine to repress CLN3 expression.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4305328&req=5

ppat.1004630.g003: The effect of inhibiting Cdc28 or the removal of Cdc28 cyclins on Fkh2 phosphorylation.Fkh2 from cells of the indicated genotype and culture condition was fractionated by 2D gels. The cdc28-as1 strain was treated with 30 µM 1NM-PP1 to inhibit Cdc28. Note it is not possible to grow cells to stationary phase with Cdc28 inhibited. The CLN3-sd strain was grown to stationary phase overnight in YEPD which allows partial de-repression of the MET3 promoter regulating CLN3 expression [76]. For hyphal and yeast growth cells were inoculated from these stationary phase cultures into YEPD medium containing 2.5 mM methionine and 0.5 mM cysteine to repress CLN3 expression.
Mentions: We next examined the Fkh2 2D profile in the cdc28-as1 mutant, and in mutants in which one of the Cdc28 G1 cyclins was either deleted (hgc1ΔΔ and ccn1ΔΔ) or down regulated using the MET3 promoter (CLN3-sd) (Fig. 3). The results showed that both inhibition of Cdc28 or lack of either Ccn1 or Cln3 prevented the shift to the hyphal profile, but there was less of an effect on the early hyphal profile in cells that lack Hgc1. Thus, the data support the conclusion that Cdc28 acts on Fkh2 early after hyphal induction, but suggest both Cln3 and Ccn1 are required to partner Cdc28. Inhibition of Cdc28 or lack of Cln3 also had a major effect on the Fkh2 profile in growing yeast cells, similar to the effect of the Fkh2(6A) mutant in these cells. Thus, while Cdc28 mediated phosphorylation is necessary for the transition to the early hyphal pattern, Fkh2 is also phosphorylated by Cdc28 during yeast growth, as would be expected from previous studies in S. cerevisiae.

Bottom Line: We confirmed that these changes in gene expression resulted in corresponding defects in pathogenic processes.Furthermore, we identified that Fkh2 interacts with the chromatin modifier Pob3 in a phosphorylation-dependent manner, thereby providing a possible mechanism by which the phosphorylation of Fkh2 regulates its specificity.Thus, we have discovered a novel cell cycle-independent phospho-regulatory event that subverts a key component of the cell cycle machinery to a role in the switch from commensalism to pathogenicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield, United Kingdom; Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore.

ABSTRACT
The opportunistic human fungal pathogen, Candida albicans, undergoes morphological and transcriptional adaptation in the switch from commensalism to pathogenicity. Although previous gene-knockout studies have identified many factors involved in this transformation, it remains unclear how these factors are regulated to coordinate the switch. Investigating morphogenetic control by post-translational phosphorylation has generated important regulatory insights into this process, especially focusing on coordinated control by the cyclin-dependent kinase Cdc28. Here we have identified the Fkh2 transcription factor as a regulatory target of both Cdc28 and the cell wall biosynthesis kinase Cbk1, in a role distinct from its conserved function in cell cycle progression. In stationary phase yeast cells 2D gel electrophoresis shows that there is a diverse pool of Fkh2 phospho-isoforms. For a short window on hyphal induction, far before START in the cell cycle, the phosphorylation profile is transformed before reverting to the yeast profile. This transformation does not occur when stationary phase cells are reinoculated into fresh medium supporting yeast growth. Mass spectrometry and mutational analyses identified residues phosphorylated by Cdc28 and Cbk1. Substitution of these residues with non-phosphorylatable alanine altered the yeast phosphorylation profile and abrogated the characteristic transformation to the hyphal profile. Transcript profiling of the phosphorylation site mutant revealed that the hyphal phosphorylation profile is required for the expression of genes involved in pathogenesis, host interaction and biofilm formation. We confirmed that these changes in gene expression resulted in corresponding defects in pathogenic processes. Furthermore, we identified that Fkh2 interacts with the chromatin modifier Pob3 in a phosphorylation-dependent manner, thereby providing a possible mechanism by which the phosphorylation of Fkh2 regulates its specificity. Thus, we have discovered a novel cell cycle-independent phospho-regulatory event that subverts a key component of the cell cycle machinery to a role in the switch from commensalism to pathogenicity.

Show MeSH
Related in: MedlinePlus