WdChs2p, a class I chitin synthase, together with WdChs3p (Class III) contributes to virulence in Wangiella (Exophiala) dermatitidis

The chitin synthase structural gene WdCHS2 was isolated by screening a subg enomic DNA library of Wangiella dermatitidis by using a 0.6-kb PCR product of the gene as a probe. The nucleotide sequence revealed a 2,784-bp open re ading frame, which encoded 928 amino acids, with a 59-by intron near its 5' end. Derived protein sequences showed highest amino acid identities with t hose derived from the CiCHS1 gene of Coccidioides immitis and the AnCHSC ge ne of Aspergillus nidulans. The derived sequence also indicated that WdChs2 p is an orthologous enzyme of Chs1p of Saccharomyces cerevisiae, which defi nes the class I chitin synthases. Disruptions of WdCHS2 produced strains th at showed no obvious morphological defects in yeast vegetative growth or in ability to carry out polymorphic transitions from yeast cells to hyphae or to isotropic forms. However, assays showed that membranes of wdchs2 Delta mutants were drastically reduced in chitin synthase activity. Other assays of membranes from a wdchs1 Delta wdchS3 Delta wdchs4 Delta triple mutant sh owed that their residual chitin synthase activity was extremely sensitive t o trypsin activation and was responsible for the majority of zymogenic acti vity. Although no loss of virulence was detected when wdchs2 Delta strains were tested in a mouse model of acute infection, wdchs2 Delta wdchs3 Delta disruptants were considerably less virulent in the same model, even though wdchs3 Delta strains also had previously shown no loss of virulence. This v irulence attenuation in the wdchs2 Delta wdchs3 Delta mutants was similarly documented in a limited fashion in more-sensitive cyclophosphamide-induced immunocompromised mice. The importance of WdChs2p and WdChs3p to the virul ence of W. dermatitidis was then confirmed by reconstituting virulence in t he double mutant by the reintroduction of either WdCHS2 or WdCHS3 into the wdchs2 Delta wdchs3 Delta mutant background.