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

Abstract

ABSTRACT By using improved transformation methods for Wangiella dermatitidis , and a cloned fragment of its chitin synthase 4 structural gene ( WdCHS4 ) as a marking sequence, the full-length gene was rescued from the genome of this human pathogenic fungus. The encoded chitin synthase product (WdChs4p) showed high homology with Chs3p of Saccharomyces cerevisiae and other class IV chitin synthases, and Northern blotting showed that WdCHS4 was expressed at constitutive levels under all conditions tested. Reduced chitin content, abnormal yeast clumpiness and budding kinetics, and increased melanin secretion resulted from the disruption of WdCHS4 suggesting that WdChs4p influences cell wall structure, cellular reproduction, and melanin deposition, respectively. However, no significant loss of virulence was detected when the wdchs4Δ strain was tested in an acute mouse model. Using a wdchs1Δ wdchs2Δ wdchs3Δ triple mutant of W. dermatitidis , which grew poorly but adequately at 25°C, we assayed WdChs4p activity in the absence of activities contributed by its three other WdChs proteins. Maximal activity required trypsin activation, suggesting a zymogenic nature. The activity also had a pH optimum of 7.5, was most stimulated by Mg 2+ , and was more inhibited by polyoxin D than by nikkomycin Z. Although the WdChs4p activity had a broad temperature optimum between 30 to 45°C in vitro, this activity alone did not support the growth of the wdchs1Δ wdchs2Δ wdchs3Δ triple mutant at 37°C, a temperature commensurate with infection.