Chitin biosynthesis in the fungal cell wall is essential for cell viability. Chitin play roles in maintaining cellular integrity and up regulation of chitin synthesis helps protect the cell from cell wall stresses or cell wall damaging antifungal agents such as caspofungin. In Candida albicans chitin is synthesised by four chitin synthases (Chs), Chs1, Chs2, Chs3, and Chs8. The biological function of Chs2 and Chs8 (class I chitin synthases) is less well understood. The major objective of this thesis was to understand the biological function of the class I chitin synthases. Chs2-YFP and Chs8-YFP showed a dynamic localisation at septation sites, which were first visualised as a bar which then contracted to a spot. This spot then separated into two spots, one on each sides of the septum. These two spots remained there until yeast cell separation, and remained at this location throughout several subsequent hyphal cell cycles. Chs2-YFP also localised to hyphal tips. The phenotype of a chs2Δchs8Δ double mutant was re-investigated using the propidium iodide. Intact dead germ tubes and hyphal tip lysis was observed in a chs2Δchs8Δ mutant cells. This suggested that Chs2 and Chs8 play a major role in the maintenance of hyphal tip integrity and polarised growth and perhaps a minor role in septum formation. Studies were also performed to assess whether phosphorylation regulated the localisation of Chs2-YFP. It was shown that the localisation of Chs2-YFP to septation sites was regulated by phosphorylation on S222. A version of Chs2-YFP that could not be phosphorylated (Chs2S222A-YFP) localised at the septa in lower amounts than the Chs2-YFP, and a version of Chs2-YFP that mimicked constitutive phosphorylation (Chs2S222E-YFP) localised normally. This suggested that phosphorylation of Chs2 on S222 facilitates the localisation of Chs2-YFP at septation sites, and that dephosphorylation is not required for this cellular localisation. In the presence of cell wall stresses (CaCl2/CFW) and caspofungin, more Chs2-YFP was observed and the average intensity of fluorescence of Chs2-YFP was higher in the presence of these stresses than in untreated cells.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:589503 |
| Date | January 2013 |
| Creators | Preechasuth, Kanya |
| Publisher | University of Aberdeen |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?pid=202781 |
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