Abstract
Candida albicans has four chitin synthases from three different enzyme classes which deposit chitin in the cell wall, including at the polarized tips of growing buds and hyphae, and sites of septation. The two class I enzymes, Chs2 and Chs8, are responsible for most of the measurable chitin synthase activity in vitro, but their precise biological functions in vivo remain obscure. In this work, detailed phenotypic analyses of a chs2Δchs8Δ mutant have shown that C. albicans class I chitin synthases promote cell integrity during early polarized growth in yeast and hyphal cells. This was supported by live cell imaging of YFP-tagged versions of the class I chitin synthases which revealed that Chs2-YFP was localized at sites of polarized growth. Furthermore, a unique and dynamic pattern of localization of the class I enzymes at septa of yeast and hyphae was revealed. Phosphorylation of Chs2 on the serine at position 222 was shown to regulate the amount of Chs2 that is localized to sites of polarized growth and septation. Independently from this post-translational modification, specific cell wall stresses were also shown to regulate the amount of Chs2 that localizes to specific sites in cells, and this was linked to the ability of the class I enzymes to reinforce cell wall integrity during early polarized growth in the presence of these stresses.
Highlights
Chitin is an essential structural polysaccharide component of cell walls and septa in fungi and is synthesized by multiple chitin synthase enzymes
We probed the biological function of the C. albicans class I chitin synthases by analyzing cell lysis phenotypes of the chs2D, chs8D and chs2Dchs8D mutants using propidium iodide (PI)
The percentage of PI-positive cells of the chs2Dchs8D double mutant was more than the sum of the percentages of PI-positive cells of the chs2D and chs8D mutants, and the deletion of CHS2 resulted in a higher proportion of PI-positive cells than the deletion of CHS8 (Table 2)
Summary
Chitin is an essential structural polysaccharide component of cell walls and septa in fungi and is synthesized by multiple chitin synthase enzymes. In Candida albicans, the most common serious fungal pathogen of humans, chitin is synthesized by a family of four isoenzymes which fall into three different classes of chitin synthase enzymes, Chs (class II), Chs (class IV), Chs and Chs (class I) (reviewed in Lenardon et al (2010b)). Together, these enzymes deposit chitin at sites of growth, which includes the polarized tips of buds and hyphae, and sites of septation. The present study shows for the first time that the C. albicans class I enzymes contribute to the protection of the nascent cell wall during polarized growth and the integrity of cells experiencing cell wall stress
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