Cloning and characterization of the chitin synthase 3 (WdCHS3) and chitin synthase 4 (WdCHS4) genes of Wangiella dermatitidis, and, expression studies of WdCHS3

Class III chitin synthases are reported to be important for hyphal growth in some filamentous fungi. Using a specific PCR product that encodes a portion of the class III chitin synthase of W. dermatitidis as a probe, the chitin synthase gene, WdCHS3, and its cDNA were isolated from this polymorphic pathogen of humans. Northern blotting showed that WdCHS3 was highly expressed under stress conditions, such as shift of cells to temperatures commensurate with infection, or to conditions of low pH, calcium limitation and nitrogen starvation, which are all known to induce cellular morphogenesis in this melanized fungus. Disruption of WdCHS3 by site-specific integrative transformation in a wild-type strain and in two temperature-sensitive morphological mutant strains, Mc3 (wdcdc2) and Hf1, did not affect the morphologies, growth rates and chitin contents of these strains at 25{dollar}spcirc{dollar}C or 37{dollar}spcirc{dollar}C, but did reduce chitin synthase activities. However, a wdchs3{dollar}Delta{dollar} mutant was significantly less virulent compared with its wild-type parental strain when yeast cells were injected into athymic and immunocompetent mice. A double disruption strain, wdchs1{dollar}Delta{dollar}wdchs3{dollar}Delta,{dollar} was also constructed using the benomyl-resistance gene as a second selective marker and the wdchs1{dollar}Delta{dollar}-2 strain. The double mutant had a phenotype similar to that of wdchs1{dollar}Delta,{dollar} but a more decreased level of chitin synthase activities.; Expression levels of WdChs3p were detected by incorporating a 6-myc tagging sequence at its N-terminus. Western blotting analysis indicated that the production of WdChs3-myc was temperature dependent and temporally regulated. Indirect immunofluorescence showed a cytoplasmic localization for WdChs3p-myc in the three predominant vegetative morphologies of W. dermatitidis. Although a GFP-tagged WdChs3p failed to make a full-size protein, it provided evidence that GFP could be expressed in W. dermatitidis and used for localizing other proteins. For more detailed characterization of WdChs3p, it was also investigated by highly expressing the WdCHS3 cDNA in Saccharomyces cerevisiae. The expressed WdChs3p shared some characteristics of other chitin synthases and was demonstrated to be a non-zymogen by substrate, UDPGlcNAc, protection when expressed in S. cerevisiae.; Taking advantage of the improving transformation technologies being developed for W. dermatitidis, and the availability of a partial fragment of WdCHS4, a marker rescue approach was employed to clone the full length chitin synthase 4 gene (WdCHS4). Sequence analysis indicated that this gene encodes a seven-transmembrane protein that was homologous with class IV chitin synthases. Disruption of WdCHS4 in the wild-type strain resulted in cells with a clumpy phenotype, a slower growth rate at 37{dollar}spcirc{dollar}C and a lower chitin content. However, the chitin synthase activities of the wdchs4{dollar}Delta{dollar} mutant did not decrease, probably due to compensating increases in the activities of other chitin synthases.; The collective results of my studies suggest that WdChs3p is a virulence factor that enriches the cell walls of W. dermatitidis with chitin under the unique and hostile stress conditions of infection and that WdChs4p serves as an auxiliary enzyme when additional chitin is required.