Wangiella Dermatitidis Research Articles

Effect of melanin and carotenoids of Exophiala (Wangiella) dermatitidis on phagocytosis, oxidative burst, and killing by human neutrophils.

The black yeast Exophiala (Wangiella) dermatitidis is an increasingly recognized pathogen and a leading cause of severe pheohyphomycosis. Melanin is thought to contribute to the virulence of E. dermatitidis. Whereas the synthesis and the redox properties of melanin have been studied intensively, the influence of melanin and carotenoids on the phagocytosis, the oxidative burst, and the killing of E. dermatitidis by human neutrophils has not been studied. To study their effects on these phenomena, we applied a combination of flow cytometry and a colony-count-dependent method. Using E. dermatitidis wild-type strain 8565 and several melanin-deficient mutants that have been described previously, we demonstrate that melanin prevents this pathogen from being killed in the phagolysosome of the neutrophils. Melanin did not influence the phagocytosis or the oxidative burst of the neutrophils involved. The carotenoids torulene and torularhodine were not found to contribute to the prevention of killing. The ability of E. dermatitidis to block the effects of the neutrophil oxidative burst may critically impair the potential of the host to sufficiently eliminate this fungal pathogen and thus may play an important role in the pathogenesis of phaeohyphomycosis.

Isolation and confirmation of function of the Coccidioides immitis URA5 (orotate phosphoribosyl transferase) gene.

The OPRTase (URA5) gene of the human pathogenic fungus, Coccidioides immitis (Ci), was cloned, sequenced, chromosome-mapped and expressed both by transformation of Escherichia coli and by complementation of wdura5Delta, an auxotrophic strain of Wangiella dermatitidis (Wd) with a disrupted URA5 gene. A functional assay of the recombinant URA5 expressed by E. coli was conducted to ensure that the isolated Ci gene encodes the appropriate enzyme. In the absence of a transformation system for Ci, we also used a reported method of introduction of heterologous DNA into cells of the phylogenetically related fungus, Wangiella dermatitidis, to confirm the function of the Ci URA5 gene. Both the genomic and cDNA sequences of the Ci URA5 gene are presented. The transcription start point and two poly(A) addition sites were confirmed. The gene contains a 714-bp ORF that translates a 238-amino-acid (aa) protein of 25.5kDa and pI of 6.5. No introns are present. The translated protein contains a single, putative N-glycosylation site. The deduced Ci protein showed 55-63% aa sequence similarity to reported fungal OPRTases. The URA5 gene was mapped to chromosome IV of Ci, and was shown to be a single copy gene by Southern and Northern hybridizations. Transformation of the wdura5Delta mutant to prototrophy was accomplished by electroporation of Wd yeast cells with the Ci URA5 gene. Cellular uptake of the heterologous DNA was confirmed by Southern hybridization. The stable transformants were unable to grow on a medium containing 5-FOA. Expression of the Ci URA5 gene can be used as a selectable marker for a transformation system, and the latter is essential for molecular studies of this pathogenic fungus.

Emendation of the genus Wangiella and a new combination, W. heteromorpha

A reexamination of the pleoanamorphic fungus, Wangiella dermatitidis, type species of Wangiella, has demonstrated the formation of phialides with distinct collarettes, annellides, acropetally produced chains of conidia and sclerotial bodies, in addition to those already ascribed, viz., phialides without collarettes and yeastlike cells. The genus is emended to accommodate all anamorphic states. A reexamination of type and authenticated material of Phialophora heteromorpha (Syn. Trichosporium heteromorphum), based on morphological characters has revealed a close relationship to W. dermatitidis. Phialophora heteromorpha is reassigned to the genus Wangiella, as W. heteromorpha. A redescription of the fungus is made.

Cloning and use of theWdURA5gene as ahisGcassette selection marker for potentially disrupting multiple genes inWangiella dermatitidis

A genomic clone encoding the Wangiella dermatitidis orotidine monophosphate pyrophosphorylase gene (WdURA5) was isolated by screening a subgenomic plasmid DNA library of this phaeohyphomycotic agent using a PCR amplification product of the gene as a probe. When plasmid DNA containing the cloned WdURA5 gene was introduced by electroporation into a wdura5 auxotrophic recipient strain derived previously by selection with 5-fluoroorotic acid (5-FOA), an apparent gene repair event occurred at high frequency without any evidence of integration of the plasmid DNA. Therefore, the hygromycin B resistance gene (the hph gene) was used as a dominant selective marker for the disruption of WdURA5 to generate a new, more stable, wdura5 auxotrophic strain. Transformation of this strain was then achieved with high efficiency and high frequency by site-specific integration using WdURA5 as a selective marker. To initiate attempts to use this marker repeatedly for multiple chitin synthase (WdCHS) gene disruptions in single strains of W. dermatitidis, a hisG_WdURA5_hisG cassette was constructed and used to disrupt WdCHS2. The WdURA5 gene in the disruptant was then successfully recycled under selection for resistance to 5-FOA.

In vitro activity of voriconazole against selected fungi

Fifty-nine isolates consisting of 14 genera and 33 species of ascomycetes, basidiomycetes, and zygomycetes were tested against amphotericin B, fluconazole, itraconazole and voriconazole using an in vitro modified macrobroth dilution procedure based upon the NCCLS M27-A standard method for yeasts. The triazoles voriconazole and itraconazole had similar MIC values, except for Acremonium alabamensis, A. strictum, Fusarium oxysporum, F. solani and Wangiella dermatitidis, which had substantially lower voriconazole MIC values. Voriconazole MIC values were lower than those for itraconazole for the 17 species of Trichosporon tested. Fluconazole had high MIC values, often greater than 128 microg ml-1.

In vitro interactions between Histoplasma capsulatum var. duboisii and other fungi.

Histoplasma capsulatum var. duboisii has only recently been shown to occur naturally in soil admixed with bat guano in a bat cave. We report our observations on the in vitro interaction of Histoplasma capsulatum var. duboisii with other fungi recovered from the same habitat or other sources by employing co-culture technique and by investigating antibiosis between H. capsulatum var. duboisii and other fungi. The range of interactions varied from total overgrowth of Histoplasma by Aspergillus fumigatus and Microsporum gypseum, through partial antagonism by Lecythophora mutabilis, to mutual inhibition by Wangiella dermatitidis. Those fungi that exhibited a competitive advantage in terms of growth did not affect the viability of Histoplasma. Lysis and mycoparasitic structures were not observed in the co-cultures. However, the cultural filtrate of Chrysosporium indicum inhibited the growth of the Histoplasma strains. The results suggest the ability of H. capsulatum var. duboisii to co-exist with a variety of other potentially pathogenic and non-pathogenic fungi in an ecological niche.

Beta-Glucosylated proteins in the cell wall of the black yeast Exophiala (Wangiella) dermatitidis.

Wild-type cells of the pathogenic black yeast Exophiala (Wangiella) dermatitidis grown in a low-pH ascorbate medium became less melanized and less resistant to Zymolyase. This was accompanied by increased staining with fluorescently labelled concanavalin A. The sugar composition of wild-type and mutant cell walls was, except for the presence of galactose, similar to that of Saccharomyces cerevisiae. Digestion of mutant cell walls with laminarinase released galactomannoproteins. In addition, the released cell wall proteins contained glucose and reacted with affinity-purified 1,6-beta-glucan antiserum, indicating that they are linked to 1,6-beta-glucan. It is proposed that 1,6-beta-glucosylated cell wall proteins generally occur among ascomycetes.

Phylogenetic analysis of ten black yeast species using nuclear small subunit rRNA gene sequences.

The nuclear small subunit rRNA genes of authentic strains of the black yeasts Exophiala dermatitidis, Wangiella dermatitidis, Sarcinomyces phaemuriformis, Capronia mansonii, Nadsoniella nigra var. hesuelica, Phaeoannellomyces elegans, Phaeococcomyces exophialae, Exophiala jeanselmei var. jeanselmei and E. castellanii were amplified by PCR and directly sequenced. A putative secondary structure of the nuclear small subunit rRNA of Exophiala dermatitidis was predicted from the sequence data. Alignment with corresponding sequences from Neurospora crassa and Aureobasidium pullulans was performed and a phylogenetic tree was constructed using the neighbor-joining method. The obtained topology of the tree was confirmed by bootstrap analysis. Based upon this analysis all fungi studied formed a well-supported monophyletic group clustering as a sister group to one group of the Plectomycetes (Trichocomaceae and Onygenales). The analysis confirmed the close relationship postulated between Exophiala dermatitidis, Wangiella dermatitidis and Sarcinomyces phaeomuriformis. This monophyletic clade also contains the telemorph species Capronia mansonii thus confirming the concept of a teleomorph connection of the genus Exophiala to a member of the herpotrichiellaceae. However, Exophiala castellanii did not belong to this clade. Therefore, this species is not the anamorph of Capronia mansonii as it was postulated.

Morphometry and stereology of the conversion of thin-walled yeasts to phase I yeast cells ofWangiella dermatitidis

Morphometric and stereological analyses of the conversion of thin-walled yeast cells to thick-walled phase I yeast cells of Wangiella dermatitidis were conducted over 15 days of growth. Log phase growth persisted for 48-60 hours and there was no loss in viability up to 15 days. Average cell volume dropped from 23 ^m3 on days 1 and 2 to 18 jiim3 on days 3 and 4, then increased to 38 jxm5 by day 15. Wide confidence limits for measurements of cell volume at each point indicated a very heterogeneous population. Wall thickness increased throughout the growth period with thickness increasing from 0.04 /um to 0.30 jj,m and the volume fraction from 0.10 to 0.24. Mitochondria and nuclei together made up less than 8% of the cell vol? ume [volume fraction of mitochondria, 0.025 (2.5%) and volume fraction of nuclei, 0.05 (5.0%)]. Volume fractions of both declined over time, as a result of increased cell volume from isotropic growth, although nuclei also may have become smaller. Glycogen was always present and increased to about 18% of the cell volume by day 4 but then decreased to 10% by day 15. Lipid was absent in early log phase cells but made up about 40% of the cell volume by day 15, and is presumably synthesized at the expense of glycogen and from residual sugar. Vacuoles, evident in early log cells (10-20% of the cell volume), declined rapidly and were rare after 8 days.

Pathogenicity of strains of the black yeast Exophiala (Wangiella) dermatitidis: an evaluation based on polymerase chain reaction.

Strains of Exophiala dermatitidis, mainly originating from patients with systemic neurotropic mycosis in Asia and from the lungs of patients with cystic fibrosis (CF) in Europe, were analysed by ribotyping of the small subunit rDNA and by random amplification of polymorphic DNA (RAPD). A characteristic banding pattern for the species was found after restriction analysis of amplified fragments V9 and ITS4. The small subunit rDNA gene of five strains was about 1800 base pairs (bp) long, while in 16 strains its length was about 3000 bp. Using RAPD, seven populations could be distinguished. European CF strains as well as Asian strains from systemic mycoses are mainly distributed over two populations, one of which contained both CF strains and a systemic strain. It is concluded that the two clinical pictures are caused by genetically similar strains. The differences in pathogenicity may be explained by immunological differences in the hosts or by differences in exposure to the fungal propagules.

Pleoanamorphic life cycle of Exophiala (Wangiella) dermatitidis.

The anamorph life cycle of the black yeast Exophiala (Wangiella) dermatitidis is described. The fungus is dimorphic, yeast cells being the prevalent form of propagation. The fungus is strongly hydrophilic, probably completing its anamorph life cycle in submersion. Adaptation to dry conditions is slow. Types of conidiogenesis comprise annellidic, phialidic and sympodial reproduction, in addition to isotropic development. Phialoconidia fail to germinate under the conditions tested, and thus may have a function other than dispersal. Sterile, multicellular bodies resembling a Capronia teleomorph are described.

New cause for false-positive results with the Pastorex Aspergillus antigen latex agglutination test

The Pastorex Aspergillus antigen test for detection of Aspergillus galactomannan antigen in the sera of patients with invasive aspergillosis is used in many clinical laboratories. A serum sample contaminated with Penicillium chrysogenum gave a strongly positive reaction (1:128) which was heat stable, was not eliminated by pronase treatment, and was not detected by a normal rabbit globulin control. This observation was shown to be due to cross-reactions of the monoclonal antibody EB-A2 used by the kit with several airborne fungi likely to contaminate serum samples, including Penicillium chrysogenum, Cladosporium herbarum, Acremonium species, Alternaria alternata, Fusarium oxysporum, Wangiella dermatitidis, and Rhodotorula rubra.

Clinical and mycological spectra of Wangiella dermatitidis infections.

The article reviews 37 cases of phaeohyphomycosis from the world literature up to 1992 accepted as being caused by Wangiella dermatitidis. The various clinical aspects of these infections are characterized, and the nomenclatural implications in the history of this emerging pathogen are discussed.

Isolation of Lecythophora mutabilis and Wangiella dermatitidis from the fruit eating bat, Eidolon helvum.

Lecythophora mutabilis was isolated from the lungs of 3 and from the liver of 2 bats, Eidolon helvum a fruit eating species. Wangiella dermatitidis was recovered from the liver of 2 bats of the same species. The isolates were pathogenic for laboratory mice when injected by subcutaneous, intraperitoneal and intravenous routes. W. dermatitidis was neurotropic in the mice injected intravenously.

Evidence for two cell division cycle (CDC) genes that govern yeast bud emergence in the pathogenic fungus Wangiella dermatitidis.

Strains Mc2 and Mc3 are morphological mutants of the melanized, pathogenic fungus Wangiella dermatitidis. These strains possess temperature-sensitive (ts) mutations designated mcm2 and mcm3, respectively. At the restrictive temperature (37 degrees C), uninucleate yeast cells of strains Mc2 and Mc3 cease budding and initiate an isotropic mode of cellular development, which is reflected in the formation of a multicellular and multinucleate morphology. Because W. dermatitidis either lacks or has an undiscovered sexual cycle, parasexual methods of analysis were used to confirm that mcm2 and mcm3 define separate bud emergence control genes in the wild-type strain. Spheroplasts of albino auxotrophs derived from strains Mc2 and Mc3 were fused and then regenerated on minimal medium. The resulting fusion products grew as darkly pigmented, prototrophic colonies. When incubated at 37 degrees C, all fusion products exhibited polarized growth predominantly as uninucleate, budding yeasts and less frequently as pseudohyphae and moniliform hyphae. Subsequent analysis of cultures derived from albino, ts segregants, which were induced from fusion products by using methyl benzimidazole-2-yl-carbamate, revealed three types of cell populations. Two resembled those expressed by strain Mc2 or Mc3. The third consisted of a cell population unlike the former, suggesting the presence of both ts mutations in all cells. These results imply that yeast development in the fusion products resulted from intergenic complementation of mcm2 and mcm3, i.e., they are nonallelic. Because mcm2 and mcm3 are equivalent to certain cdc lesions in the yeast Saccharomyces cerevisiae, we have renamed the analogous genes defined by the mutations in W. dermatitidis as CDC1 and CDC2. To our knowledge, these are the first CDC genes identified in a dematiaceous fungus.

Fungal melanonychia: ungual phaeohyphomycosis caused by Wangiella dermatitidis

A 51-year-old female Japanese patient developed black pigmentation affecting both big toe-nails. Direct potassium hydroxide examination of the nail tissue demonstrated clusters of spherical dematiaceous cells, toruloid hyphae, and septate hyphae. Wangiella dermatitidis was repeatedly isolated from the affected toe-nail lesions. This case represents the first documented case of ungual phaeohyphomycosis, 'fungal melanonychia,' caused by the dematiaceous fungus W. dermatitidis. The patient was successfully treated with a topical solution of bifonazole.

Exoantigen test for the rapid identification ofExophiala spinifera

Exophiala spinifera, one of the etiologic agents of subcutaneous phaeohyphomycosis, may be wrongly identified as Exophiala jeanselmei because of the morphologic similarity of the two species. A reagent containing a precipitin specific for E. spinifera was produced by absorbing antiserum to E. spinifera with E. jeanselmei serotype 1 antigen. Using this absorbed antiserum, we were able to correctly identify isolates of E. spinifera and differentiate them from those of Exophiala alcalophila, E. jeanselmei (serotypes 1, 2 and 3), Exophiala moniliae, Exophiala pisciphila, Exophiala salmonis, Phaeoannellomyces werneckii and Wangiella dermatitidis.

Experimental phaeohyphomycosis

The authors performed an experimental infection of the rabbit eye with Wangiella dermatitidis which had been isolated from the corneal ulcer of a patient. The fungus was inoculated into the front chamber and the vitreous body. The disease showed a trend to spontaneous recovery. The individual phases of the experimental infection were followed by histology and electronmicroscopy, both TEM and SEM. Different stages of development of the polymorphous fungus, as sclerotic bodies, mycelial filaments and yeast-like cells, could be demonstrated.

A previously undescribed synanamorph ofWangiella dermatitidis

A black yeast isolated from a Korean patient with subcutaneous phaeohyphomycosis was studied and identified as Wangiella dermatitidis (Kano) McGinnis on the basis of its distinctive morphology, and physiologic and serologic characteristics. In addition to the characteristic diagnostic features of W. dermatitidis, we encountered unusual multicellular structures. These consisted of globose endoconidia contained within thick-walled, dematiaceous bodies. Mycologic observation indicated that a cell increased in size, developed a thickened outer cell wall, and subsequently developed internal septa that split or lysed down their central portions to separate the developing endoconidia. To our knowledge, this reproductive form has not been previously associated with W. dermatitidis. We describe this new synanamorph of the highly polymorphic black fungus W. dermatitidis.

Corneal Phaeohyphomycosis Caused by <i>Wangiella dermatitidis</i>

A case is described of a corneal abscess of the left eye of a patient, male, 35 years, from which there was found in pure culture a mycotic micro-organism, Exophiala (Wangiella) dermatitidis, determined also in the native preparation from the pus. The patient is feeble-minded, suffers from von Recklinghausen's disease and has an impairment of the immune system (above all the reduction of T lymphocytes).