Regulation of chitin synthesis in Candida albicans
The study of the regulation of chitin synthesis in the pathogen Candida albicans is a challenging field. It not only offers a possibility of a better understanding of the dimorphic transition but also may help in the development of an effective antifungal. The suggestion that the regulation of cell wall synthesis may be closely coupled to the turgor pressure of the fungus has been investigated. The synthesis of chitin by the enzyme chitin synthase was studied under conditions of osmotic stress. Mixed membrane fractions from protoplasts of C.albicans incubated in medium of low osmolality exhibited up to four-fold greater native enzyme activity as compared to protoplasts incubated at high osmolality. This was also the case for preparations from whole cells of C.albicans, Coprinus cinereus and Saccharomyces cerevisiae and also from protoplasts from S.cerevisiae. Trypsin-treated enzyme preparations did not show this regulation to the same degree. The addition of nikkomycin Z, a differential chitin synthase inhibitor, partially restored this regulation. The synthesis of chitin, assessed by following the incorporation of (14C)-GlcNAc into chitin in the cell wall, was also greater in C.albicans cells incubated in medium of low osmolality. However, the incorporation of (14C)-GlcNAc into the cell wall of regenerating protoplasts exhibited the opposite effect. This was substantiated further by measuring the fluorescence of regenerating protoplasts following the addition of Calcofluor white. Following the attempted cloning of the C.albicans CHS3 (CSD2) gene, a detailed northern analysis of three chitin synthase genes during growth and dimorphism of C.albicans was performed. CHS1 was expressed during both the yeast and hyphal phases of growth while CHS2 and CHS3 were preferentially expressed in the hyphal form. There was no difference in expression of the chitin synthase genes in invasive and non-invasive clinical isolates of C.albicans and all three genes showed highest levels of mRNA when grown in medium of neutral pH.