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  current news   Press   selected story    
     
  6 February 2015  
  Cell Cycle-Independent Phospho-Regulation of Fkh2 during Hyphal Growth Regulates Candida albicans Pathogenesis
 
 




Authors
Jamie A. Greig1,2, Ian M. Sudbery3, Jonathan P. Richardson4, Julian R. Naglik4, Yue Wang2,5,* & Peter E. Sudbery1,*

1  Department of Molecular Biology and Biotechnology, University of Sheffield, Western Bank, Sheffield,    United Kingdom
2 Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore
3  Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, United Kingdom
4  Mucosal and Salivary Biology Division, King’s College London Dental Institute, King’s College London,    London, United Kingdom
5  Department of Biochemistry, Yong Loo Ling School of Medicine, National University of Singapore,    Singapore

*Corresponding authors

Published in PLOS Pathogens 24 January 2015.

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.

Figure:

Figure Legend: 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.

For more information on Yue WANG's laboratory, please click here.