Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 165-188). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Texas at Austin, 1998. / Vita. Includes bibliographical references (leaves 190-217). Available also in a digital version from Dissertation Abstracts.

Carotenoid pigments in W. dermatitidis, the first pathogenic, dematiaceous fungus in which carotenoid pigments nave been reported, are located primarily (81%) in lipid organelles which floated on the surface of the supernatant fraction of lysed cells. Pigment in this fraction could be extracted with ethyl ether without prior treatment with acetone indicating the pigment is unbound in the lipid organelle. Eight percent remains after exhaustive ether extraction and is recovered after the sample is treated with acetone indicating this fraction is non-covalently bound to proteins in the membranes associated with the lipid organelle. The remaining pigment (about 12%) represents contamination of the supernatant with the lipid organelles.

Carotenoids in W. dermatitids were found to be associated with membranes of lipid globules and/or proteins dispersed in the lipids in the yeast-phase of the organism. The lipid globules increase in size and the pigment concentrations increase with age of the cell. Electron micrographs show these organelles to be surrounded by a single unit membrane. The free carotenoids are extractable with ethyl ether from pigmented fractions of osmotically ruptured protoplasts only after the sample has been treated with acetone, indicating the pigment is non-covalently bound, presumably to a protein.

Not available / text

To study the function of WdStuAp and WdPacCp in Wangiella dermatitidis, a black, polymorphic fungal pathogen of humans with yeast phase predominance, WdSTUA and WdPACC were cloned, sequenced, disrupted and expressed. WdStuAp was most similar to the APSES proteins of Aspergillus species and its APSES DNA-binding domain was located in its N-terminal half. Deletion of WdSTUA in W. dermatitidis induced convoluted instead of normal smooth colony surface growth on the rich, yeast maintenance agar medium, YPDA, at 37°C. Additionally, deletion of WdSTUA repressed aerial hyphal growth, conidiation and invasive hyphal growth on the nitrogen poor, hyphae-inducing agar medium, PDA, at 25°C. Ectopic expression of WdSTU Arepressed the convoluted colony surface growth on YPDA at 37°C, and also strongly repressed hyphal growth on PDA at 25°C and 37°C. Expression of WdSTUA in S. cerevisiae induced pseudohyphal growth on the nitrogen poor medium. WdPacCp was also most similar to the PacCp proteins of Aspergillus species. Three zinc finger DNA-binding motifs were at the N-terminus, and the C-terminus had the signaling protease cleavage site. WdPACC was more expressed at neutral-alkaline pH than at acidic pH. Truncation of the coding sequence for about 40 residues upstream of the conserved processing protease cleavage site of WdPacCp affected growth on YPDA, increased sensitivity to Na⁺ stress, decreased growth level at neutral-alkaline pH, and repressed hyphal growth on PDA at 25°C. Truncation of the coding sequence for the conserved signaling protease box of WdPacCp impaired growth and reduced RNA expression of class II chitin synthase gene WdCHS1 at acidic pH, and activated hyphal growth on PDA. My results suggested that WdStuAp and WdPacCp play important roles in yeast-hyphal transitions in W. dermatitidis, and that WdPacCp is particularly important for W. dermatitidis to adapt to different ambient pH conditions.

Thesis (Ph. D.)--University of Texas at Austin, 2003. / Vita. Includes bibliographical references. Available also from UMI Company.

Thesis (Ph. D.)--University of Texas at Austin, 2007. / Vita. Includes bibliographical references.

Septins are a highly conserved family of eukaryotic proteins having significant homology within and among species. In the budding yeast, Saccharomyces cerevisiae, a septin-based hierarchy of proteins is required to localize chitin in the bud neck prior to septum formation. However, this process has not been clarified in a filamentous, conidiogenous fungus capable of yeast growth, such as Wangiella dermatitidis, a polymorphic agent of human phaeohyphomycosis. Prior studies of this melanized mold showed that some chitin synthase mutants (wdchsΔ) have defects in yeast septum formation, suggesting that the septins of W. dermatitidis might functionally associate with some of its chitin synthases (WdChsp). To test this hypothesis, four vegetative septin homologs of S. cerevisiae were cloned from W. dermatitidis and designated WdCDC3, WdCDC10, WdCDC11, and WdCDC12. Of the four, only WdCDC3 functionally complemented completely a strain of S. cerevisiae with a ts mutation in the corresponding gene, although WdCDC12 did so partially. Functional characterizations by mutagenesis of the four W. dermatitidis septin genes revealed that resulting mutants (wdcdc[delta]) each had unique defects in yeast growth and morphology, indicating that each septin carried out a distinct function. Furthermore, when a wdcdc10[delta] mutation was introduced into five different wdchs[delta] strains, weak genetic interactions were detected between WdCDC10 and WdCHS3 and WdCHS4, and a strong interaction between and WdCHS5. Cytological studies showed that WdChs5p was mislocalized in some septin mutants, including wdcdc10[delta]. These results confirmed that in W. dermatitidis septins are important for proper cellular morphogenesis, cytokinesis, and especially septum formation through associations with some chitin synthases. / text

Morphological transitions in Wangiella dermatitidis, a causative agent of human phaeohyphomycosis, influence virulence processes in this polymorphic fungus. My project first involved the cloning and characterizion of the β(1→3) glucan synthase gene WdFKS1, which encodes the enzyme's catalytic subunit, followed by cloning and characterizing the WdRHO1 gene, which encodes its regulatory subunit. To better understand the Rho-type GTPase-mediated regulation of cell polarity and its role in fungal morphological transitions, a homologue of WdRAC1 from a W. dermatitidis was subsequently identified by degenerate PCR and gene walking. Gene deletions of WdFKS1 and WdRHO1 in haploid W. dermatitidis were lethal, whereas the deletion of WdRAC1 was not. RNA interference on WdFKS1 mRNA expression resulted in incomplete septa and damaged cell wall integrity, as well as slow growth rate in W. dermatitidis. Overexpression studies, after site-specific integrations of WdRHO1 and WdRAC1 alleles under control of the glaA promoter into the nonessential WdPKS1 locus, showed the different alleles had different effects on the cell morphological development. For example, whereas overexpression of the wdrho1⁺ allele did not affect the growth rate of W. dermatitidis, the overexpression of wdrho1[superscript G14V], a constitutively active mutation, slowed growth and repressed true filamentous hyphal growth by promoting pseudohyphal growth. While the deletion of WdRAC1 did not affect growth, its loss retarded polarized hyphal growth in a hyphal-inducing minimal medium. Moreover, three new phenotypes of a previously derived WdCDC42 deletion mutant were discovered during this study: in the first, the wdcdc42[Delta] mutant displayed cell lysis when incubated in YPMaltose medium at 37°C; in the second, a dark budding neck abnormality was found after Calcoflour staining; and in the third, the wdcdc42[Delta] mutant displayed no branching during true hyphal growth. Interestingly, the overexpression of wdrac1[superscript G16V] complemented the second and the third phenotypes caused by the WdCDC42 deletion. In addition, the wdcdc42[Delta]/wdrac1[superscript G16V] double mutant unexpectedly displayed an interrupted carotenogenesis pathway. These results support that in W. dermatitidis, Rho-type GTPases play essential roles in growth rate determination and cellular morphogenesis, especially while producing polarized hyphal growth during its many morphological transitions. / text