Coccidioides posadasii Contains a Single 1,3-β-Glucan Synthase Gene That Appears To Be Essential for Growth

Coccidioides species (C. immitis and C. posadasii) are primary fungal pathogens that cause disease by the respiratory route in humans and many other species of mammals. The genus has recently been reorganized from one species (C. immitis) into two species, with C. posadasii being localized to Texas, Arizona, Mexico, and South America whereas C. immitis is found only in the area of the San Joaquin Valley of California (13). Although the species are genetically distinct, differences in morphology or pathogenicity have not yet been delineated. Coccidioides spp. are dimorphic and produce characteristic structures for the saprobic and parasitic growth phases (for a review of the life cycle, see reference 6). The soil-dwelling saprobic phase is mycelial, with asexual spores produced through septation of hyphae and subsequent differentiation of alternate cellular compartments into arthroconidia, which contain on average two nuclei but sometimes contain more (6). Mature conidia 3 to 6 by 2 to 4 μm in size are liberated by physical disruption and disperse readily in the wind. Upon inhalation into the host lung, the arthroconidium undergoes a phase transition to form a large (60 to >100 μm) spherule that undergoes internal septation to produce hundreds of endospores. Most infections are relatively mild and resolve without specific treatment, although about 30% of infections produce flu-like symptoms (39). However, some infections are severe and require extensive antifungal therapy (40). The fungal cell wall is conceptually attractive as a target for antifungal therapies. It is critical for maintaining cell shape and integrity and contains the polysaccharides 1,3-β-glucan and chitin (4, 38), which are not constituents of mammalian cells. Antifungal agents that act by inhibiting their synthesis could be highly selective. 1,3-β-glucan is the predominant polysaccharide in most fungal cell walls, including those of Saccharomyces cerevisiae, Candida albicans, and Neurospora crassa (14, 38), and is a significant component in the walls of C. posadasii in all growth phases (21, 42). The glucan synthase complex has been best studied in S. cerevisiae and consists of a catalytic subunit encoded by either of two genes (FKS1 or FKS2) and a regulatory subunit (RHO1). The FKS genes encode large polypeptides (∼200 kDa) with 16 membrane-spanning domains that synthesize 1,3-β-glucan from UDP-glucose subunits (4, 11, 33). Rho1p is a small GTPase that in its prenylated form interacts with Fksp catalytic subunits to activate 1,3-β-glucan synthase (22, 23, 32, 37). Glucan synthase activity is essential in yeast. An FKS1 FKS2 double deletion is lethal, although single deletions are viable, suggesting overlapping functions of the two catalytic polypeptides (24, 33). FKS2 is required for sporulation, and a third gene in yeast, FKS3, is nonessential for growth or sporulation (41). FKS1 homologs have been reported in the pathogenic fungi C. albicans, Cryptococcus neoformans, Paracoccidioides brasiliensis, and Pneumocystis carinii and the filamentous fungi Aspergillus nidulans and Aspergillus fumigatus (3, 10, 28, 35, 41). The FKS1 genes have been demonstrated to be essential single-copy genes in C. albicans, C. neoformans, and A. fumigatus (3, 10, 41). The echinocandins and pneumocandins are a class of antifungal drugs that inhibit 1,3-β-glucan synthase (9). Use of one echinocandin, caspofungin (Merck Pharmaceuticals), is successful in the treatment of infections due to Candida spp. and Aspergillus spp. and has been approved by the Food and Drug Administration for such treatment. However, it has not been effective against C. neoformans (2, 25, 29, 36). Caspofungin has been shown to be effective in treating mice infected with Coccidioides spp. (19), but relatively high doses were required, and in earlier studies a different echinocandin, cilofungin, was found not to be useful (17). Although these results suggested that 1,3-β-glucan synthase might not be a therapeutically important antifungal target in Coccidioides spp., other pharmacologic factors could be responsible for these marginal effects. To investigate 1,3-β-glucan synthase as an antifungal target in Coccidioides spp., we sought to identify the gene and disrupt the function of the FKS homolog(s) in C. posadasii. We have identified a single gene, FKS1, with similarity to other fungal FKS genes, and disruption experiments suggest that FKS1 is essential for growth of C. posadasii.