WdChs4p, a Homolog of Chitin Synthase 3 in Saccharomyces cerevisiae, Alone Cannot Support Growth of Wangiella(Exophiala) dermatitidis at the Temperature of Infection

Chitin, a linear molecule of β-(1-4)-linked N-acetylglucosamine, is a major structural constituent of the fungal cell wall (16, 49). Chitin quantity varies in different fungi and in different cell types of the same fungus (3, 8, 59). It is a minor (1%) component of cell walls of most yeasts, but amounts are usually higher in the cell walls of filamentous fungi (14). In Saccharomyces cerevisiae, more than 90% of the chitin is located in the region of the yeast cell bud scar (38). However, greater amounts of mislocalized chitin occur in a variety of cell division cycle mutants of S. cerevisiae, indicating that chitin synthesis is temporally and spatially regulated during the yeast cell cycle (44, 47). In vegetative hyphae of filamentous fungi, chitin deposition is most concentrated in septa and at the apexes of growing hyphae (25, 59). Chitin synthases are responsible for the polymerization of chitin and are primarily zymogens associated with fungal plasma membranes (12). In both S. cerevisiae and Candida albicans, three chitin synthase structural genes (CHS) have been identified and characterized (10, 37). Numerous other CHS genes have been identified in other fungi. Based on derived amino acid sequences of PCR products, fungal chitin synthases were first grouped as three classes (7). Additional classes were then defined, and now at least five isozyme classes are recognized (33). However, only in the case of S. cerevisiae has each isozyme (Chs) been linked with some certainty to specific functions during cell growth and development (9, 11, 51, 57). In this fungus, Chs3p, which belongs to the class IV group of chitin synthases (7), produces 90% of the cell wall chitin in yeast cells budding normally, and this chitin is localized both in the lateral wall and in the chitin ring that serves as the bud emergence locus (57). Although no single Chs enzyme is essential for the viability of S. cerevisiae, strains without functional class II (Chs2p) and class IV (Chs3p) isozymes lose viability (11, 16). In contrast to this central role for the class IV enzyme in S. cerevisiae, in filamentous fungi the class III chitin synthases, which have no homolog in yeast, are most often reported to be essential for normal hyphal growth (34, 61, 62). In fact, the class IV chitin synthases identified in Neurospora crassa, Aspergillus nidulans, and A. fumigatus have been regarded as redundant enzymes (1, 5, 40), even though additional genes encoding class IV chitin synthase homologs were found in both Aspergillus species (33, 35, 52). Wangiella (Exophiala) dermatitidis is an asexual fungal pathogen of humans that is associated with cutaneous and subcutaneous phaeohyphomycosis (30). In vivo, this member of the Fungi Imperfecti produces a variety of vegetative growth forms with dark, melanized (dematiaceous) cell walls, such as ovoid yeasts, pseudohyphae, hyphae and isotropically enlarged, spherical cells, and multicellular forms (20, 32, 41). In vitro, yeast cells of W. dermatitidis are easily manipulated in ways that allow morphological transitions to isotrophic multicellular forms and hyphae (18, 24, 28, 54). This inherent vegetative polymorphism of W. dermatitidis allows it to serve as a valuable model for the study of the more than 100 other dematiaceous fungi known to cause human infection (31, 55). The numerous new transformation and gene disruption systems developed for the molecular manipulation of this organism (31, 45, 64) also make it an unusually attractive model for determining the function of potential cell wall-related virulence factors, such as chitin, in dimorphic and polymorphic pathogenic fungi. In yeast cells of W. dermatitidis, chitin is found in relatively low amounts and is localized primarily in septal regions (19, 26, 54, 56). Multicellular forms and hyphae also have chitin in their septa but have considerable additional chitin in other cell wall areas (54–56). Because these morphological transitions are accompanied not only by increases in chitin but also increases in cell wall 1,8-dihydroxynaphthalene melanin (19, 54), melanin biosynthesis has been studied extensively and shown to contribute significantly to the pathogenicity and virulence of W. dermatitidis (21–23, 48). However, the specific role of chitin and its contribution to virulence have not been similarly established for this pathogen. Three different genes (WdCHS) encoding class I, II, and III chitin synthases were initially identified in W. dermatitidis by PCR and Southern blotting and then cloned by screening genomic and cDNA libraries (7, 31, 36, 56, 58, 63). In this study, we describe the cloning, characterization, and effects of disruption of the fourth WdCHS gene (WdCHS4) of W. dermatitidis, and we also characterize the enzymatic activity of its gene product (WdChs4p). Our results showed that this gene is most likely constitutively expressed, that its product is grouped to class IV, and that the chitin contributed by WdChs4p influences wall structure, cell surface properties, cellular reproduction, and melanization but not virulence. Our results also showed that WdChs4p could not alone support growth at 37°C of a triple chitin synthase mutant of W. dermatitidis having only an intact WdCHS4 gene. However, because this triple mutant grew adequately at 25°C, we were able to characterize the activity of the class IV-type isozyme of W. dermatitidis in the absence of any confounding influence of its other WdChs activities. Furthermore, our comparative studies of the wild-type strain with strains having only WdCHS4 disrupted and with the triple mutant derived by the sequential disruption of WdCHS1, WdCHS2, and WdCHS3 but not WdCHS4, allowed us to suggest why WdChs4p of W. dermatitidis may not be particularly important to virulence.