WdChs2p, a Class I Chitin Synthase, Together with WdChs3p (Class III) Contributes to Virulence in Wangiella (Exophiala) dermatitidis

摘要

The chitin synthase structural gene WdCHS2 was isolatedby screening a subgenomic DNA library of Wangielladermatitidis by using a 0.6-kb PCR product of the gene as aprobe. The nucleotide sequence revealed a 2,784-bp open reading frame,which encoded 928 amino acids, with a 59-bp intron near its 5′ end.Derived protein sequences showed highest amino acid identities withthose derived from the CiCHS1 gene of Coccidioidesimmitis and the AnCHSC gene of Aspergillusnidulans. The derived sequence also indicated that WdChs2p is anorthologous enzyme of Chs1p of Saccharomyces cerevisiae,which defines the class I chitin synthases. Disruptions ofWdCHS2 produced strains that showed no obviousmorphological defects in yeast vegetative growth or in ability to carryout polymorphic transitions from yeast cells to hyphae or to isotropicforms. However, assays showed that membranes of wdchs2Δmutants were drastically reduced in chitin synthase activity. Otherassays of membranes from awdchs1Δwdchs3Δwdchs4Δ triplemutant showed that their residual chitin synthase activity wasextremely sensitive to trypsin activation and was responsible for themajority of zymogenic activity. Although no loss of virulence wasdetected when wdchs2Δ strains were tested in a mousemodel of acute infection, wdchs2Δwdchs3Δdisruptants were considerably less virulent in the same model, eventhough wdchs3Δ strains also had previously shown no lossof virulence. This virulence attenuation in thewdchs2Δwdchs3Δ mutants was similarlydocumented in a limited fashion in more-sensitivecyclophosphamide-induced immunocompromised mice. The importance ofWdChs2p and WdChs3p to the virulence of W. dermatitidis wasthen confirmed by reconstituting virulence in the double mutant by thereintroduction of either WdCHS2 or WdCHS3 intothe wdchs2Δwdchs3Δ mutant background.