Molecular regulation of chitin synthesis in Candida albicans
The aim of this project was to construct multiple chitin synthase (chs) mutants of Candida albicans sin order to elucidate the function of the Class I enzymes, Chs2p and Chs8p. The single Class I mutants created by Gow et al. (1994), Mio et al. (1996), and Munro et al. (2003) did not have a major phenotype that could indicate a function. The main phenotype for both mutants was a reduction in chitin synthase activity, 80% reduction in chs2Delta/chs2Delta mutant (Munro et al., 1998) and 30% reduction in the chs8Delta/chs8Delta mutant (Munro et al., 2003). A double chs2/chs2 chs8Delta/chs8Delta mutant was created in this project and the mutant phenotype analysed. The mutant was indistinguishable from wild type except for chitin synthase activity, which was dramatically reduced to 3% of wild type activity in yeast cells, and 1% wild type activity in hyphal cells. These two class I enzymes therefore contribute 97-99% of chitin synthase activity measured in vitro. Further combinations of deletion mutants were also created to further investigate the role of specific chitin synthases in cell wall synthesis and cellular growth. The chitin microfibril architecture of this mutant and other available chs mutants was examined in order to determine whether each Chs protein was responsible for synthesizing a particular form of chitin. Wild type cells had long microfibrils visible at the septum and short crystalline fibrils in the cell wall. Analyses of all available chs mutants revealed that Chs8p synthesis was required for all of the long microfibrils at the septum as well as in the lateral cell wall, and Chs3p synthesised the majority of the short crystalline fibrils. From investigations with the chs1Delta/chs1Delta mutant it was clear that Chs1p contributed chitin to the septum (Munro et al.,2001). Chs1p apparently also contributes to the formation of long microfibrils in the cell wall. Chs2p appeared to synthesise a small amount of short crystalline fibrils in the cell wall. In conclusion each Chsp appears to synthesise a specific form of chitin that together are assembled at particular cellular locations to make the mature cell wall and septum.