A Conserved Machinery Underlies the Synthesis of a Chitosan Layer in the Candida Chlamydospore Cell Wall

Abstract

The polysaccharide chitosan is found in the cell wall of specific cell types in a variety of fungal species where it contributes to stress resistance, or in pathogenic fungi, virulence. Under certain growth conditions, the pathogenic yeast Candida dubliniensis forms a cell type termed a chlamydospore, which has an additional internal layer in its cell wall compared to hyphal or yeast cell types. We report that this internal layer of the chlamydospore wall is rich in chitosan. The ascospore wall of Saccharomyces cerevisiae also has a distinct chitosan layer. As in S. cerevisiae, formation of the chitosan layer in the C. dubliniensis wall requires the chitin synthase CHS3 and the chitin deacetylase CDA2 In addition, three lipid droplet-localized proteins-Rrt8, Srt1, and Mum3-identified in S. cerevisiae as important for chitosan layer assembly in the ascospore wall are required for the formation of the chitosan layer of the chlamydospore wall in C. dubliniensis These results reveal that a conserved machinery is required for the synthesis of a distinct chitosan layer in the walls of these two yeasts and may be generally important for incorporation of chitosan into fungal walls.IMPORTANCE The cell wall is the interface between the fungal cell and its environment and disruption of cell wall assembly is an effective strategy for antifungal therapies. Therefore, a detailed understanding of how cell walls form is critical to identify potential drug targets and develop therapeutic strategies. This study shows that a set of genes required for the assembly of a chitosan layer in the cell wall of S. cerevisiae is also necessary for chitosan formation in a different cell type in a different yeast, C. dubliniensis Because chitosan incorporation into the cell wall can be important for virulence, the conservation of this pathway suggests possible new targets for antifungals aimed at disrupting cell wall function.

Keywords: cell wall; chitin deacetylase; chitin synthase; chlamydospore; lipid droplet.

FIG 1

Effect of different carbon sources on the chlamydospore formation. A wild-type C. dubliniensis strain (Cd1465) was spotted on synthetic agar medium containing the indicated carbon sources and photographed on agar after 24 h of growth. Gal, galactose; GlcNAc, N-acetylglucosamine; GlcN, glucosamine. White arrows highlight examples of chlamydospores. Scale bar, 50 nm.

FIG 2

Fluorescence analysis of the chlamydospore wall of C. dubliniensis. (A) Chlamydospores of WT (Cd1465) and cda2Δ (BEM7) strains were stained with Eosin Y to visualize the chitosan layer and imaged using a GFP filter set. Wild-type (WT) chlamydospores with no Eosin Y staining are shown as control. (B) WT (Cd1465), dit1Δ (BEM9), or dit2Δ (BEM10) strains were grown on SG medium to induce chlamydospores and then visualized by differential interference contrast (DIC) or fluorescence microscopy using a dityrosine filter set (excitation [Ex.], 320 nm; emission [Em.], 410 nm). Arrowheads indicate examples of chlamydospores visible in the images. Scale bar, 10 μm.

FIG 3

Fluorescence analysis of the C. albicans chlamydospore wall. (A) Unstained chlamydospores of C. albicans strain NLC1 were imaged using either a dityrosine filter set (Ex., 320 nm; Em., 410 nm) or a DAPI filter set (Ex., 375 nm; Em., 475 nm). (B) Chlamydospores of NLC1 stained with Eosin Y and imaged using a GFP filter set. Arrowheads indicate examples of chlamydospores visible in the images. Scale bar, 10 μm.

FIG 4

Effect of mutations in C. dubliniensis orthologs of S. cerevisiae spore wall genes on the chlamydospore wall. (A) Cells of strains of the indicated genotype were grown on SGlycerol medium and then stained with both Eosin Y to label chitosan and Calcofluor White (CFW) to label chitin or chitosan. Arrowheads indicate examples of chlamydospores visible in the images. Arrows indicate examples of CFW-stained septa. Scale bar, 10 μm. (B) The intensity of the Eosin Y fluorescence was categorized as bright, dim, or no fluorescence for each chlamydospore, and the number of chlamydospores in each category for each strain was quantified. For each strain, the value represents the average for 100 chlamydospores in each of three independent experiments. Error bars indicate one standard deviation. One asterisk (*) indicates significant difference at P < 0.05; two asterisks (**) indicates significant difference at P < 0.0005 (Student t test).

FIG 5

Complementation of the chitosan defect by the wild-type alleles. (A) A wild-type copy of CHS3, CDA2, MUM3, or SRT1 gene, respectively, was integrated into the corresponding deletion mutant (strains BEM15 to BEM18). Cells were grown on SGlycerol medium, and Eosin Y staining of chlamydospores with or without reintroduction of the wild-type allele was examined. DIC, differential interference contrast. Scale bar, 10 μm. (B) Rescue of Eosin Y staining by the wild-type alleles was quantified as in Fig. 4B..

FIG 6

Electron microscopy of the chlamydospore wall of C. dubliniensis. (A) Chlamydospores were induced, and cells of different strains were stained with osmium-thiocarbohydrazide: WT (CD1465), cda2Δ (BEM7), chs3Δ (BEM8), mum3Δ (BEM11), srt1Δ (BEM14), and rrt8Δ (BEM13). For each strain, a pair of images is shown. The lower image is a higher magnification of the boxed region in upper image. Arrowheads indicate the inner cell wall layer. (B) Quantification of the thickness of the chitosan layer in each strain. Data represented are the means of measurements from 20 chlamydospores. The thickness of the chitosan layer was measured at 5 different positions on each chlamydospore. Error bars indicate one standard deviation. One asterisk (*) indicates a significant difference at P < 0.00005; two asterisks (**) in indicates P < 5E–10 (Student t test). Scale bar, 500 nm.

FIG 7

Lipid droplets in chlamydospores. WT cells (CD1465) growing on SGlucose or SGlycerol medium or the indicated cda2Δ (BEM7), chs3Δ (BEM8), mum3Δ (BEM11), srt1Δ (BEM14), and rrt8Δ (BEM13) mutant strains grown on SGlycerol were stained with MDH to label lipid droplets and visualized using a BFP filter. Scale bar, 10 μm.

FIG 8

Localization of Cda2, Mum3, Rrt8, and Srt1 in chlamydospores. (A) Eosin Y staining of chlamydospores in WT (CD1465) mum3Δ MUM3-yEMRFP (BEM20) and srt1Δ SRT1-yEmRFP (BEM22) strains was quantified as in Fig. 4B. (B) WT (Cd1456) cells expressing no RFP fusion or strains expressing different MUM3-, SRT1-, or RRT8-yEmRFP fusions (BEM20, -21, or -22) were grown on SGlycerol medium, stained with MDH, and visualized through both BFP and RFP filters. Scale bar, 10 μm.

FIG 9

Model for organization of the C. dubliniensis chlamydospore and S. cerevisiae ascospore walls. The organization of the different layers of the walls is shown with respect to the cell plasma membrane. The linkages between components are based on the known linkages in the vegetative cell wall of S. cerevisiae (25). The nature of the cross-links within and between the chitosan and dityrosine layers is unknown.