Molecular analysis of chitin synthesis in Candida albicans
The aim of this project as to construct a chsI null mutant strain of Candida albicans and analyse the mutant phenotype in order to elucidate the function of CaChs1p. The inability to generate such a strain by conventional gene disruption suggested CaCHSI was important or essential for growth. This was verified by creating a conditional chsI mutant strain (KWC340) by placing the only wild type copy of CaCHSI under the control of a regulatable maltase (MRPI) promoter. When strain KWC340 was grown in conditions that were repressing for the MRPI promoter CaCHSI mRNA was not detected by Northern analysis and cells grew as multinucleate chains. Calcoflour staining indicated these cells lacked a primary septum and this was confirmed by examining thin sections by electron microscopy. WGA-gold labelling of thin sections of three chs mutants of C. albicans confirmed that CaChs1p synthesises chitin of the primary septum, CaChs3p synthesises lateral cell wall chitin and the chitin ring at the site of bud emergence whereas chitin localisation of the chs2 mutant was indistinguishable from wild type cells. All three chs mutants formed chlamydospores. Measurement of chitin synthase activity in vitro indicated that CaChs2p encodes the major enzyme activity. This activity was similar to ScChs1p in respect to nikkomycin sensitivity, zymogenicity and preference for divalent cations suggesting Cachs2p is the functional homologue of ScChs1p. This project addressed the conflicting reports on the chitin content of chs2 mutant hyphal cells. Chitin contents were measured in three ways. The deacetylation/deamination protocol of Ride & Drysdale used by Gow et al. (1994) showed a 44% reduction in hyphal chitin in the chs2 mutant. Measurement of total glucosamine by acid hydrolysis as employed by Mio et al. (1996) determined wild type chitin levels in chs2 hyphae as did the third method which measures GlcNAC released from cells by the action of two enzymes, a chitinase and N-acetylglucosaminidase.