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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.

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Fkh2 is differentially phosphorylated between yeast and hyphal growth.A) Early G1 cells expressing Fkh2-YFP were collected by elutriation and re-inoculated into yeast growth conditions. Samples were taken for αGFP Western blot to observe Fkh2 phosphorylation and microscopy to follow cell cycle progression via budding and DAPI stained nuclei (n = 50) Note YFP is recognised by the αGFP monoclonal antibody; αCdc11 was used as a control for equal loading. B) Early G1 cells expressing Fkh2-YFP and Cdc12-mCherry were collected by elutriation and re-inoculated into hyphal growth conditions. Samples were taken as above, with cell cycle progression followed by monitoring septin ring formation and nuclear migration/division (n = 50). C) Confirmation of Fkh2 phosphorylation by phosphatase treatment. 80 min yeast and 40 min hyphae samples were taken and lysates treated at 30°C for 1 h with/without Lambda-phosphatase (NEB) and then resolved by 7% 1D PAGE. D) Fkh2 phosphorylation early on hyphal induction. Samples were taken at the indicated time points after hyphal induction and resolved by 1D PAGE as previously mentioned. In Figs. 1B–D αPSTAIRE was used as the loading control. E) Fkh2-YFP was isolated from cells in the culture conditions and times indicated and fractionated by 2D gel electrophoresis. Note the region of darkening at the acidic edge of the gel is where the sample was applied and does not come from Fkh2. An intensity profile is shown above each autoradiograph. In this and subsequent figures the profile was scaled to give maximum height to the maximum peak in the informative part of the gel. Where necessary some values from the non-specific part of the gel were omitted. Fig. 1E is shown with an independent replicate in S2 Fig.
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ppat.1004630.g001: Fkh2 is differentially phosphorylated between yeast and hyphal growth.A) Early G1 cells expressing Fkh2-YFP were collected by elutriation and re-inoculated into yeast growth conditions. Samples were taken for αGFP Western blot to observe Fkh2 phosphorylation and microscopy to follow cell cycle progression via budding and DAPI stained nuclei (n = 50) Note YFP is recognised by the αGFP monoclonal antibody; αCdc11 was used as a control for equal loading. B) Early G1 cells expressing Fkh2-YFP and Cdc12-mCherry were collected by elutriation and re-inoculated into hyphal growth conditions. Samples were taken as above, with cell cycle progression followed by monitoring septin ring formation and nuclear migration/division (n = 50). C) Confirmation of Fkh2 phosphorylation by phosphatase treatment. 80 min yeast and 40 min hyphae samples were taken and lysates treated at 30°C for 1 h with/without Lambda-phosphatase (NEB) and then resolved by 7% 1D PAGE. D) Fkh2 phosphorylation early on hyphal induction. Samples were taken at the indicated time points after hyphal induction and resolved by 1D PAGE as previously mentioned. In Figs. 1B–D αPSTAIRE was used as the loading control. E) Fkh2-YFP was isolated from cells in the culture conditions and times indicated and fractionated by 2D gel electrophoresis. Note the region of darkening at the acidic edge of the gel is where the sample was applied and does not come from Fkh2. An intensity profile is shown above each autoradiograph. In this and subsequent figures the profile was scaled to give maximum height to the maximum peak in the informative part of the gel. Where necessary some values from the non-specific part of the gel were omitted. Fig. 1E is shown with an independent replicate in S2 Fig.

Mentions: To identify further targets of Cdc28 in hyphal development, we first identified C. albicans proteins which contain a cluster of the consensus Cdc28 target motifs, S/TPxK/R (x, any amino acid). We then used a band shift assay in one dimensional polyacrylamide gel electrophoresis (1D PAGE) to determine whether any of these proteins were differentially phosphorylated in hyphae compared to yeast. In addition to the proteins described in the introduction we identified changes in the phosphorylation profile of Orf19.3469 (S1 Fig.), a possible homolog of the S. cerevisiae Stb1 protein that regulates the MBF transcription at START [42], Orf19.1948 (S1 Fig.), a protein of unknown function, and Fkh2 which is known to play a key role in cell cycle progression (S1 Fig.). Here we report our analysis of Fkh2 phosphorylation and its cell-cycle independent role in promoting the expression of genes involved in pathogenesis. Fig. 1 presents an experiment where early G1 yeast cells expressing Fkh2-YFP were collected by elutriation and then reinoculated either into yeast growth conditions (YEPD and 30°C, pH 4.0) or hyphal growth conditions (YEPD plus 10% serum and 37°C, pH 7.0). In yeast growth conditions, the appearance of small buds, large buds and binucleate cells was recorded and plotted against time (Fig. 1A). In hyphal cells we plotted germ tube emergence, the appearance of a septin ring within the germ tube as visualized by Cdc12-mCherry fluorescence, nuclear migration as visualised by DAPI staining, and the appearance of binucleate cells (Fig. 1B). A change in the phosphorylation profile of Fkh2 was indicated by the appearance of a double band and the disappearance of the slower migrating band upon phosphatase treatment (Fig. 1C). (Note in Fig. 1A the septin Cdc11 was used as a loading control whereas in Fig. 1B the loading control was Cdc28/Pho85 identified by a monoclonal anti-PSTAIRE antibody). In yeast cells, the Fkh2-YFP band became double after the appearance of small buds (Fig. 1A), consistent with phosphorylation in S-phase as previously documented in S. cerevisiae [43]; this then collapsed to one band when the cells became bi-nucleate. In contrast, Fkh2 was present as a double band from 20–60 min after hyphal induction, well before the appearance of the septin ring (Fig. 1B), which marks the start of the cell cycle [44]. Cdc28, partnered by the cyclin Ccn1, and in conjunction with the Gin4 kinase, has been shown to phosphorylate the septin Cdc11 within 5 min of hyphal induction [30]. To determine if Fkh2 is similarly targeted at this early stage, we repeated the experiment collecting samples at 5-min intervals after hyphal induction. Fkh2 showed an additional retarded band after 5 min (Fig. 1D). Thus, whereas Fkh2 is phosphorylated in S-phase in yeast cells, it is rapidly phosphorylated upon hyphal induction in a cell cycle-independent fashion.


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)

Fkh2 is differentially phosphorylated between yeast and hyphal growth.A) Early G1 cells expressing Fkh2-YFP were collected by elutriation and re-inoculated into yeast growth conditions. Samples were taken for αGFP Western blot to observe Fkh2 phosphorylation and microscopy to follow cell cycle progression via budding and DAPI stained nuclei (n = 50) Note YFP is recognised by the αGFP monoclonal antibody; αCdc11 was used as a control for equal loading. B) Early G1 cells expressing Fkh2-YFP and Cdc12-mCherry were collected by elutriation and re-inoculated into hyphal growth conditions. Samples were taken as above, with cell cycle progression followed by monitoring septin ring formation and nuclear migration/division (n = 50). C) Confirmation of Fkh2 phosphorylation by phosphatase treatment. 80 min yeast and 40 min hyphae samples were taken and lysates treated at 30°C for 1 h with/without Lambda-phosphatase (NEB) and then resolved by 7% 1D PAGE. D) Fkh2 phosphorylation early on hyphal induction. Samples were taken at the indicated time points after hyphal induction and resolved by 1D PAGE as previously mentioned. In Figs. 1B–D αPSTAIRE was used as the loading control. E) Fkh2-YFP was isolated from cells in the culture conditions and times indicated and fractionated by 2D gel electrophoresis. Note the region of darkening at the acidic edge of the gel is where the sample was applied and does not come from Fkh2. An intensity profile is shown above each autoradiograph. In this and subsequent figures the profile was scaled to give maximum height to the maximum peak in the informative part of the gel. Where necessary some values from the non-specific part of the gel were omitted. Fig. 1E is shown with an independent replicate in S2 Fig.
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ppat.1004630.g001: Fkh2 is differentially phosphorylated between yeast and hyphal growth.A) Early G1 cells expressing Fkh2-YFP were collected by elutriation and re-inoculated into yeast growth conditions. Samples were taken for αGFP Western blot to observe Fkh2 phosphorylation and microscopy to follow cell cycle progression via budding and DAPI stained nuclei (n = 50) Note YFP is recognised by the αGFP monoclonal antibody; αCdc11 was used as a control for equal loading. B) Early G1 cells expressing Fkh2-YFP and Cdc12-mCherry were collected by elutriation and re-inoculated into hyphal growth conditions. Samples were taken as above, with cell cycle progression followed by monitoring septin ring formation and nuclear migration/division (n = 50). C) Confirmation of Fkh2 phosphorylation by phosphatase treatment. 80 min yeast and 40 min hyphae samples were taken and lysates treated at 30°C for 1 h with/without Lambda-phosphatase (NEB) and then resolved by 7% 1D PAGE. D) Fkh2 phosphorylation early on hyphal induction. Samples were taken at the indicated time points after hyphal induction and resolved by 1D PAGE as previously mentioned. In Figs. 1B–D αPSTAIRE was used as the loading control. E) Fkh2-YFP was isolated from cells in the culture conditions and times indicated and fractionated by 2D gel electrophoresis. Note the region of darkening at the acidic edge of the gel is where the sample was applied and does not come from Fkh2. An intensity profile is shown above each autoradiograph. In this and subsequent figures the profile was scaled to give maximum height to the maximum peak in the informative part of the gel. Where necessary some values from the non-specific part of the gel were omitted. Fig. 1E is shown with an independent replicate in S2 Fig.
Mentions: To identify further targets of Cdc28 in hyphal development, we first identified C. albicans proteins which contain a cluster of the consensus Cdc28 target motifs, S/TPxK/R (x, any amino acid). We then used a band shift assay in one dimensional polyacrylamide gel electrophoresis (1D PAGE) to determine whether any of these proteins were differentially phosphorylated in hyphae compared to yeast. In addition to the proteins described in the introduction we identified changes in the phosphorylation profile of Orf19.3469 (S1 Fig.), a possible homolog of the S. cerevisiae Stb1 protein that regulates the MBF transcription at START [42], Orf19.1948 (S1 Fig.), a protein of unknown function, and Fkh2 which is known to play a key role in cell cycle progression (S1 Fig.). Here we report our analysis of Fkh2 phosphorylation and its cell-cycle independent role in promoting the expression of genes involved in pathogenesis. Fig. 1 presents an experiment where early G1 yeast cells expressing Fkh2-YFP were collected by elutriation and then reinoculated either into yeast growth conditions (YEPD and 30°C, pH 4.0) or hyphal growth conditions (YEPD plus 10% serum and 37°C, pH 7.0). In yeast growth conditions, the appearance of small buds, large buds and binucleate cells was recorded and plotted against time (Fig. 1A). In hyphal cells we plotted germ tube emergence, the appearance of a septin ring within the germ tube as visualized by Cdc12-mCherry fluorescence, nuclear migration as visualised by DAPI staining, and the appearance of binucleate cells (Fig. 1B). A change in the phosphorylation profile of Fkh2 was indicated by the appearance of a double band and the disappearance of the slower migrating band upon phosphatase treatment (Fig. 1C). (Note in Fig. 1A the septin Cdc11 was used as a loading control whereas in Fig. 1B the loading control was Cdc28/Pho85 identified by a monoclonal anti-PSTAIRE antibody). In yeast cells, the Fkh2-YFP band became double after the appearance of small buds (Fig. 1A), consistent with phosphorylation in S-phase as previously documented in S. cerevisiae [43]; this then collapsed to one band when the cells became bi-nucleate. In contrast, Fkh2 was present as a double band from 20–60 min after hyphal induction, well before the appearance of the septin ring (Fig. 1B), which marks the start of the cell cycle [44]. Cdc28, partnered by the cyclin Ccn1, and in conjunction with the Gin4 kinase, has been shown to phosphorylate the septin Cdc11 within 5 min of hyphal induction [30]. To determine if Fkh2 is similarly targeted at this early stage, we repeated the experiment collecting samples at 5-min intervals after hyphal induction. Fkh2 showed an additional retarded band after 5 min (Fig. 1D). Thus, whereas Fkh2 is phosphorylated in S-phase in yeast cells, it is rapidly phosphorylated upon hyphal induction in a cell cycle-independent fashion.

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