Expression of a Constitutively Active Cdc42 Homologue Promotes Development of Sclerotic Bodies but Represses Hyphal Growth in the Zoopathogenic Fungus Wangiella ( Exophiala ) dermatitidis

Abstract

In contrast to the CDC42 homologues of Saccharomyces cerevisiae and Schizosaccharomyces pombe, the WdCDC42 gene in the human pathogenic fungus Wangiella (Exophiala) dermatitidis was found to be nonessential for cell viability. Expression of the constitutively active allele wdcdc42 G14V at 37°C induced nonpolarized growth that led to cell enlargement and multiple nucleation. The swollen cells subsequently converted into planate divided bicellular forms or multiply septated sclerotic bodies in post-log phase, when the G14V-altered protein was diminished. The wdcdc42 G14V mutation also strongly repressed filamentous growth both in the wild-type strain and in the temperature-sensitive hyphal-form mutant Hf1. In contrast, overexpression of the dominant negative alleles wdcdc42 T19N and wdcdc42 D120A had no obvious effect on fungal-cell polarization. These results suggested that WdCdc42p plays a unique regulatory role in cellular morphogenesis in W. dermatitidis. Activation of this protein in response to extracellular or intracellular signals seems to commit its yeast-like cells to a phenotype transition that produces sclerotic bodies while repressing hyphal development.

The CDC42 Homolog of the Dimorphic Fungus Penicillium marneffei Is Required for Correct Cell Polarization during Growth but Not Development

“…The equivalent CDC42 G14V mutation in E. dermatitidis results in a phenotype similar to that of P. marneffei, resulting in nonpolarized growth that leads to cell enlargement, multiple nucleation, and repressed hyphal growth. However, unlike what was found for P. marneffei, the dominant-negative mutation (cdc42 D120A ) was not found to affect fungal cell polarization (40). The phenotypes of the P. marneffei dominant-activated and -negative mutations are also similar to those produced by the equivalent mutations in S. pombe, which both give rise to enlarged, round, or misshapen cells which, in addition, can be abnormally elongated (27).…”

“…To confirm the positions of the predicted introns, reverse transcriptase PCR was performed on poly(A) mRNA produced from a vegetative mycelium grown at 25°C and the PCR product was sequenced. Database searches and sequence comparisons show that the cflA gene is most closely related to the homologous gene from Exophiala dermatitidis (AF162788.1 [40]) which is 83% identical at the DNA level and 96% identical in predicted protein sequence (Fig. 1).…”

WdChs1p, a class II chitin synthase, is more responsible than WdChs2p (Class I) for normal yeast reproductive growth in the polymorphic, pathogenic fungus Wangiella (Exophiala) dermatitidis

“…Procedures for staining of the cell wall with Calcofluor (Sigma) and for the staining of nuclei with DAPI (4′,6′-diamide-2-phenylendole; Accurate Chemical), as well as those for transmission electron microscopy, have also been described (Cooper et al 1984;Ye and Szaniszlo 2000).…”

Molecular cloning and characterization of WdTUP1, a gene that encodes a potential transcriptional repressor important for yeast-hyphal transitions in Wangiella (Exophiala) dermatitidis

“…The procedures for staining cell wall chitin with Calcofluor (Sigma) or for staining nuclei with DAPI (4′, 6′-diamidino-2-phenylindole; Accurate Chemical, USA) have been described previously (Ye and Szaniszlo, 2000). Colony images were recorded using a Cannon G3 digital camera attached to an Olympus Model SZ dissecting microscope (Olympus Corporation, Japan).…”

“…When required, agar (1.5% w/v) was added to produce solid medium (YPDA) for strain maintenance and mutant selection. The potato dextrose agar (PDA), and cornmeal agar (CMA) were both purchased from Difco Scientific (USA), whereas the CDNA-starch agar and the CDN and SD agars (CDNA & SDA, respectively) and broths (CDNB & SDB, respectively) were prepared as described previously (Cooper and Szaniszlo, 1993;Karuppayil and Szaniszlo, 1997;Ye and Szaniszlo, 2000). E. coli XL1-blue (Stratagen, USA) was grown in Luria-Bertani medium supplemented with 100 μg/ml ampicillin and was used for the subcloning and plasmid preparation.…”