RNA interference of WdFKS1 mRNA expression causes slowed growth, incomplete septation and loss of cell wall integrity in yeast cells of the polymorphic, pathogenic fungus Wangiella (Exophiala) dermatitidis.

As one of the main components of the fungal cell wall, ß-1,3-glucan provides the mechanical strength to protect fungal protoplasts. The enzyme responsible for the synthesis of ß-1,3-glucans in fungi is ß-1,3-glucan synthase. Here we report the cloning, sequencing and characterization of the WdFKS1 gene, which in the pathogenic fungus Wangiella dermatitidis encodes the catalytic domain of its ß-1, 3-glucan synthase. Because our research suggested that WdFKS1 is a single copy essential gene, we used RNA interference to reduce its expression. Reduction of the WdFKS1 messenger retarded growth and caused the loss of cell wall integrity of yeast cells, but not hyphae or sclerotic cells. We suggest that the WdFKS1 in this polymorphic agent of phaeohyphomycosis is not only required for cell wall construction and maintenance, but also is involved in septum formation.

Roles of the pH signaling transcription factor PacC in Wangiella (Exophiala) dermatitidis.

To study the function of the PacC transcription factor in Wangiella dermatitidis, a black, polymorphic fungal pathogen of humans with yeast-phase predominance, the PACC gene was cloned, sequenced, disrupted and expressed. Three zinc finger DNA-binding motifs were found at the N-terminus, and a signaling protease cleavage site at the C-terminus. PACC was more expressed at neutral-alkaline pH than at acidic pH. Truncation at about 40 residues of the coding sequence upstream of the conserved protease processing cleavage site of PacC affected growth on a nutrient-rich medium, increased sensitivity to Na(+) stress, decreased yeast growth at neutral-alkaline pH, and repressed hyphal growth on a nutrient-poor medium at 25 degrees C. Truncation at the coding sequence for the conserved signaling protease box of PacC impaired growth and reduced RNA expression of the class II chitin synthase gene at acidic pH. The results suggested that PacC is important not only for the adaptation of W. dermatitidis to different ambient pH conditions and Na(+) stress conditions, but also for influencing yeast-hyphal transitions in this agent of phaeohyphomycosis.

Cytolocalization of the class V chitin synthase in the yeast, hyphal and sclerotic morphotypes of Wangiella (Exophiala) dermatitidis.

Wangiella (Exophiala) dermatitidis is a polymorphic fungus that produces polarized yeast and hyphae, as well as a number of non-polarized sclerotic morphotypes. The phenotypic malleability of this agent of human phaeohyphomycosis allows detailed study of its biology, virulence and the regulatory mechanisms responsible for the transitions among the morphotypes. Our prior studies have demonstrated the existence of seven chitin synthase structural genes in W. dermatitidis, each of which encodes an isoenzyme of a different class. Among them, the class V chitin synthase (WdChs5p) is most unique in terms of protein structure, because it has an N-terminal myosin motor-like domain with a P-loop (MMD) fused to its C-terminal chitin synthase catalytic domain (CSCD). However, the exact role played by WdChs5p in the different morphotypes remains undefined beyond the knowledge that it is the only single chitin synthase required for sustained cell growth at 37 degrees C and consequently virulence. This report describes the expression in Escherichia coli of a 12kDa polypeptide (WdMyo12p) of WdChs5p, which was used to raise in rabbits a polyclonal antibody that recognized exclusively its MMD region. Results from the use of the antibody in immunocytolocalization studies supported our previous findings that WdChs5p is critically important at infection temperatures for maintaining the cell wall integrity of developing yeast buds, elongating tips of hyphae, and random sites of expansion in sclerotic forms. The results also suggested that WdChs5p localizes to the regions of cell wall growth in an actin-dependent fashion.

New biosynthetic step in the melanin pathway of Wangiella (Exophiala) dermatitidis: evidence for 2-acetyl-1,3,6,8-Tetrahydroxynaphthalene as a novel precursor.

The predominant cell wall melanin of Wangiella dermatitidis, a black fungal pathogen of humans, is synthesized from 1,8-dihydroxynaphthalene (D2HN). An early precursor, 1,3,6,8-tetrahydroxynaphthalene (T4HN), in the pathway leading to D2HN is reportedly produced directly as a pentaketide by an iterative type I polyketide synthase (PKS). In contrast, the bluish-green pigment in Aspergillus fumigatus is produced after the enzyme Ayg1p converts the PKS product, the heptaketide YWA1, to T4HN. Previously, we created a new melanin-deficient mutant of W. dermatitidis, WdBrm1, by random molecular insertion. From this strain, the altered gene WdYG1 was cloned by a marker rescue strategy and found to encode WdYg1p, an ortholog of Ayg1p. In the present study, two gene replacement mutants devoid of the complete WdYG1 gene were derived to eliminate the possibility that the phenotype of WdBrm1 was due to other mutations. Characterization of the new mutants showed that they were phenotypically identical to WdBrm1. Chemical analyses of mutant cultures demonstrated that melanin biosynthesis was blocked, resulting in the accumulation of 2-acetyl-1,3,6,8-tetrahydroxynaphthalene (AT4HN) and its oxidative product 3-acetylflaviolin in the culture media. When given to an albino W. dermatitidis strain with an inactivated WdPKS1 gene, AT4HN was mostly oxidized to 3-acetylflaviolin and deacetylated to flaviolin. Under reduced oxygen conditions, cell-free homogenates of the albino converted AT4HN to D2HN. This is the first report of evidence that the hexaketide AT4HN is a melanin precursor for T4HN in W. dermatitidis.

Immunoaffinity purification of the class V chitin synthase of Wangiella (Exophiala) dermatitidis.

The class V chitin synthase is unique because it has a myosin motor-like domain fused to its catalytic domain. The biochemical properties of this enzyme and its function remain undefined beyond the knowledge that it is the only single chitin synthase required for sustained cell growth at elevated temperatures and, consequently, virulence. This report describes our successful efforts to isolate and purify an active and soluble form of the enzyme from the cell membranes of Wangiella by using a specific polyclonal antibody. To our knowledge, this is the first purification of a single chitin synthase of a filamentous fungus.

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

The general transcriptional repressor Tup1p is known to influence cell development in many fungi. To determine whether the Tup1p ortholog (WdTup1p) of Wangiella dermatitidis also influences cellular development in this melanized, polymorphic human pathogen, the gene (WdTUP1) that encodes this transcription factor was isolated, sequenced and disrupted. Phylogenetic analysis showed that the WdTup1p sequence was closely related to homologues in other polymorphic, conidiogenous fungi. Disruption of WdTUP1 produced mutants (wdtup1Delta) with pronounced growth and cellular abnormalities, including slow growth on various agar media and exclusively as a filamentous morphotype in liquid media. We concluded that WdTup1p represents an important switch regulator that controls the yeast-to-filamentous growth transition. However, detailed observations of the filamentous growth of the disruption mutant showed that the hyphae produced by the wdtup1Delta mutants, unlike those of the wild-type, were arrested at a stage prior to the formation of true hyphae and subsequent conidia production.

Transcription and expression analyses of WdCHS5, which encodes a class V chitin synthase with a myosin motor-like domain in Wangiella (Exophiala) dermatitidis.

WdChs5p in Wangiella dermatitidis is a class V chitin synthase that is required for sustained cell growth at the temperature of infection (37 degrees C) and its encoding gene, WdCHS5, has a differential expression feature. Nuclear run-on and mRNA stability assays showed that increased WdCHS5 mRNA synthesis was the major factor responsible for the increased WdCHS5 transcript at 37 degrees C. Epitope tagging of WdChs5p in W. dermatitidis showed that the WdChs5p-myc protein had a differential expression feature that was similar to the differential transcription of the WdCHS5 gene. In conclusion, it is shown that transcriptional regulation is the first and probably the most important control point of the expression of WdCHS5.

Effects of disrupting the polyketide synthase gene WdPKS1 in Wangiella [Exophiala] dermatitidis on melanin production and resistance to killing by antifungal compounds, enzymatic degradation, and extremes in temperature.

BACKGROUND: Wangiella dermatitidis is a human pathogenic fungus that is an etiologic agent of phaeohyphomycosis. W. dermatitidis produces a black pigment that has been identified as a dihydroxynaphthalene melanin and the production of this pigment is associated with its virulence. Cell wall pigmentation in W. dermatitidis depends on the WdPKS1 gene, which encodes a polyketide synthase required for generating the key precursor for dihydroxynaphthalene melanin biosynthesis. RESULTS: We analyzed the effects of disrupting WdPKS1 on dihydroxynaphthalene melanin production and resistance to antifungal compounds. Transmission electron microscopy revealed that wdpks1Delta-1 yeast had thinner cell walls that lacked an electron-opaque layer compared to wild-type cells. However, digestion of the wdpks1Delta-1 yeast revealed small black particles that were consistent with a melanin-like compound, because they were acid-resistant, reacted with melanin-binding antibody, and demonstrated a free radical signature by electron spin resonance analysis. Despite lacking the WdPKS1 gene, the mutant yeast were capable of catalyzing the formation of melanin from L-3,4-dihyroxyphenylalanine. The wdpks1Delta-1 cells were significantly more susceptible to killing by voriconazole, amphotericin B, NP-1 [a microbicidal peptide], heat and cold, and lysing enzymes than the heavily melanized parental or complemented strains. CONCLUSION: In summary, W. dermatitidis makes WdPKS-dependent and -independent melanins, and the WdPKS1-dependent deposition of melanin in the cell wall confers protection against antifungal agents and environmental stresses. The biological role of the WdPKS-independent melanin remains unclear.

Quantification of homogentisate-1,2-dioxygenase expression in a fungus degrading ethylbenzene.

A quantitative real time reverse transcription polymerase chain reaction (qRT-PCR) assay was utilized to quantify the expression of ElHDO in the fungus Exophiala lecanii-corni during the biodegradation of ethylbenzene and other volatile organic pollutants. The assay was applied to measure the impact of pollutant mixtures on ElHDO expression relative to that of a housekeeping gene (18S rRNA). Three compounds were tested in mixtures with ethylbenzene: methyl propyl ketone, phenylacetate and o-xylene. These chemicals repressed, induced, or had no effect on ethylbenzene degradation, respectively. The results demonstrate that the gene target expression value (T(N)) is a useful parameter for evaluating the effect of pollutant mixtures on gene expression. T(N) was found to reflect macroscopic changes in ethylbenzene utilization rates although these two parameters were not related in a linear fashion for all compounds. The assay was log-linear over 5 orders of magnitude of RNA concentration and reproducible between samples (the largest T(N) standard deviation was 20%). The comparative qRT-PCR assay used in this research represents a viable alternative to absolute quantification methods to monitor in situ fungal gene expression in natural and engineered environmental systems.

Comparative genomic and transcriptomic analysis of wangiella dermatitidis, a major cause of phaeohyphomycosis and a model black yeast human pathogen.

Black or dark brown (phaeoid) fungi cause cutaneous, subcutaneous, and systemic infections in humans. Black fungi thrive in stressful conditions such as intense light, high radiation, and very low pH. Wangiella (Exophiala) dermatitidis is arguably the most studied phaeoid fungal pathogen of humans. Here, we report our comparative analysis of the genome of W. dermatitidis and the transcriptional response to low pH stress. This revealed that W. dermatitidis has lost the ability to synthesize alpha-glucan, a cell wall compound many pathogenic fungi use to evade the host immune system. In contrast, W. dermatitidis contains a similar profile of chitin synthase genes as related fungi and strongly induces genes involved in cell wall synthesis in response to pH stress. The large portfolio of transporters may provide W. dermatitidis with an enhanced ability to remove harmful products as well as to survive on diverse nutrient sources. The genome encodes three independent pathways for producing melanin, an ability linked to pathogenesis; these are active during pH stress, potentially to produce a barrier to accumulated oxidative damage that might occur under stress conditions. In addition, a full set of fungal light-sensing genes is present, including as part of a carotenoid biosynthesis gene cluster. Finally, we identify a two-gene cluster involved in nucleotide sugar metabolism conserved with a subset of fungi and characterize a horizontal transfer event of this cluster between fungi and algal viruses. This work reveals how W. dermatitidis has adapted to stress and survives in diverse environments, including during human infections.

WdStuAp, an APSES transcription factor, is a regulator of yeast-hyphal transitions in Wangiella (Exophiala) dermatitidis.

APSES transcription factors are well-known regulators of fungal cellular development and differentiation. To study the function of an APSES protein in the fungus Wangiella dermatitidis, a conidiogenous and polymorphic agent of human phaeohyphomycosis with yeast predominance, the APSES transcription factor gene WdSTUA was cloned, sequenced, disrupted, and overexpressed. Analysis showed that its derived protein was most similar to the APSES proteins of other conidiogenous molds and had its APSES DNA-binding domain located in the amino-terminal half. Deletion of WdSTUA in W. dermatitidis induced convoluted instead of normal smooth colony surface growth on the rich yeast maintenance agar medium yeast extract-peptone-dextrose agar (YPDA) at 37 degrees C. Additionally, deletion of WdSTUA repressed aerial hyphal growth, conidiation, and invasive hyphal growth on the nitrogen-poor, hypha-inducing agar medium potato dextrose agar (PDA) at 25 degrees C. Ectopic overexpression of WdSTUA repressed the convoluted colony surface growth on YPDA at 37 degrees C, and also strongly repressed hyphal growth on PDA at 25 degrees C and 37 degrees C. These new results provide additional insights into the diverse roles played by APSES factors in fungi. They also suggest that the transcription factor encoded by WdSTUA is both a positive and negative morphotype regulator in W. dermatitidis and possibly other of the numerous human pathogenic, conidiogenous fungi capable of yeast growth.

Relative gene expression quantification in a fungal gas-phase biofilter.

Monitoring of gas-phase biofilter performance generally relies on macroscale measurements that neglect the molecular level phenomena that can control the biodegradation process. The present study was undertaken to determine whether or not quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) could detect changes in relative gene expression resulting from feed variations typically encountered in the field. Specifically, homogentisate-1,2-dioxygenase, ElHDO, expression was quantified as a function of short-term chemical feed variations and shutdown period in a biofilter seeded with a pure culture of the fungus Exophiala lecanii-corni. ElHDO was previously shown to be involved in ethylbenzene degradation in E. lecanii-corni. Overall, relative gene target expression numbers (T(N)) were consistent with gas-phase biofilter performance during each short-term experiment although no direct mathematical correlation was found between T(N) and ethylbenzene removal rate. During the chemical feed experiments, no effect on T(N) was measured in the presence of o-xylene which does not affect ElHDO expression. In the presence of phenylacetate, an inducer of ElHDO, T(N) increased once a threshold substrate concentration was exceeded. When methyl propyl ketone, a repressor of ElHDO, was introduced, T(N) decreased rapidly and acted as a leading indicator of bioreactor failure. In the transient loading experiments, ElHDO expression slowly decreased over a 24-h time period when the ethylbenzene feed was discontinued, but rapidly recovered upon its re-introduction. These results indicate that qRT-PCR reflects microbial activity changes that occur in gas-phase biofilters in response to short-term changes in feed conditions and provides a useful complement to the macroscale measurements typically collected.

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.

The chitin synthase gene WdCHS1 was isolated from a partial genomic DNA library of the pathogenic polymorphic fungus Wangiella dermatitidis. Sequencing showed that WdCHS1 encoded a class II chitin synthase composed of 988 amino acids. Disruption of WdCHS1 produced strains that were hyperpigmented in rich media, grew as yeast at wild-type rates at both 25 and 37 degrees C and were as virulent as the wild type in a mouse model. However, detailed morphological and cytological studies of the wdchs1Delta mutants showed that yeast cells often failed to separate, tended to be enriched with chitin in septal regions and, sometimes, were enlarged with multiple nuclei, had broader mother cell-daughter bud regions and had other cell wall defects seen considerably less often than in the wild type or wdchs2 Delta strains. Disruption of WdCHS1 and WdCHS2 in the same background revealed that WdChs1p had functions synergistic to those of WdChs2p, because mutants devoid of both isozymes produced growth that was very abnormal at 25 degrees C and was not viable at 37 degrees C unless osmotically stabilized. These results suggested that WdChs1p was more responsible than WdChs2p for normal yeast cell reproductive growth because strains with defects in the latter exhibited no morphological abnormalities, whereas those with defects in WdChs1p were frequently impaired in one or more yeast developmental processes.

Identification of a homogentisate-1,2-dioxygenase gene in the fungus Exophiala lecanii-corni: analysis and implications.

Exophiala lecanii-corni is a dimorphic fungus capable of degrading several volatile organic compounds (VOCs) including ethylbenzene, which has been classified as a hazardous air pollutant by the Environmental Protection Agency. In contrast to bacterial species, little is known about the mechanisms of fungal degradation of VOCs. The results described herein suggest a potential pathway for ethylbenzene degradation in E. lecanii-corni via styrene, phenylacetate and homogentisate. Consistent with this proposed pathway, a full-length homogentisate-1,2-dioxygenase gene (ElHDO) has been identified, cloned and sequenced. The nucleotide sequence of ElHDO consists of a 1,452-bp open reading frame encoding a protein with 484 amino acids. The expression of the gene product increases when grown on ethylbenzene, further suggesting that it could be involved in ethylbenzene degradation and may be responsible for the aromatic ring cleavage reaction. In addition, a 907-bp fragment isolated upstream from this gene shares 78% sequence identity at the amino acid level with the amino acid sequences of two fungal phenylacetate hydroxylase genes. This observation suggests that the genes responsible for ethylbenzene degradation may be clustered. This research constitutes the first step towards a better understanding of ethylbenzene degradation in E. lecanii-corni.

Molecular genetic studies of the model dematiaceous pathogen Wangiella dermatitidis.

The rapidly improving molecular genetic tractability of Wangiella (Exophiala) dermatitidis significantly enhances its usefulness as a model for the more than 100 other dematiaceous (melanized) agents of human disease. Previously this model was based almost exclusively on its vegetative polymorphism, which at the simplest level is expressed as three well-characterized modes of growth (e.g., blastic, apical and isotropic) that produce myriad yeast, hyphal and sclerotic phenotypes. This cellular plasticity is important for a dematiaceous model pathogen because some are hyphal in nature but exist almost exclusively as sclerotic bodies in infected tissue, whereas others are hyphal both in nature and in tissue, and still others exist in nature predominantly as yeast, but as mixtures of yeast, hyphae and sclerotic bodies in tissue. By exploiting the polymorphism of W. dermatitidis, any phenotype of another dematiaceous pathogen can be produced for study of the regulation of its development and its contribution to pathogenicity and virulence. The coupling of this asset with the recent finding that its haploid, uninucleate yeast cell is easily transformed molecularly, and the even more recent development of systems for both random and targeted gene disruptions and for site-specific, integrative gene overexpression studies suggest that it will continue as the preferred model for the dematiaceous fungi and irrespective of the mycosis involved. The results reviewed here aim to confirm this contention, stimulate others to study this fungus, and demonstrate that W. dermatitidis is exceptionally useful for discovering by molecular genetic techniques cell wall-associated virulence factors in fungi, and in particular in the dematiaceous fungi.

Compensatory expression of five chitin synthase genes, a response to stress stimuli, in Wangiella (Exophiala) dermatitidis, a melanized fungal pathogen of humans.

Numerous chitin synthase structural (CHS) genes have been identified in fungi, and usually there are several CHS genes per species. Compensatory expression of one CHS gene in response to defects in other CHS genes has not been reported. Five chitin synthase structural (WdCHS) genes have been identified in the melanized human pathogen Wangiella dermatitidis: WdCHS1, WdCHS2, WdCHS3, WdCHS4 and WdCHS5. This study showed that increased WdCHS expression existed as a compensatory mechanism in response to stress induced by chitin synthase gene disruptions, or by exposure of the wild-type or two temperature-sensitive morphological mutants, for short or long periods, to 37 degrees C. In general, the compensatory responses varied with each WdCHS gene, and in accordance with the hypothesized functions of the chitin synthase (WdChsp) encoded. It is suggested that these compensatory responses indicate that WdCHS gene transcription in W. dermatitidis functions as part of a cell-wall integrity pathway in a manner similar to that recently described for Saccharomyces cerevisiae.

Characterization of two polyketide synthase genes in Exophiala lecanii-corni, a melanized fungus with bioremediation potential.

Exophiala lecanii-corni has significant bioremediation potential because it can degrade a wide range of volatile organic compounds. In order to identify sites for the insertion of genes that might enhance this potential, a genetic analysis of E. lecanii-corni was undertaken. Two polyketide synthase genes, ElPKS1 and ElPKS2, have now been discovered by a PCR-based strategy. ElPKS1 was isolated by a marker rescue technique. The nucleotide sequence of ElPKS1 consists of a 6576-bp open reading frame encoding a protein with 2192 amino acids, which was interrupted by a 60-bp intron near the 5' end and a 54-bp intron near the 3' end. Sequence analysis, results from disruption experiments, and physiological tests showed that ElPKS1 encoded a polyketide synthase required for melanin biosynthesis. Since ElPKS1 is non-essential, it is a desirable bioengineering target site for the insertion of native and foreign genes. The successful expression of these genes could enhance the bioremediation capability of the organism. ElPKS2 was cloned by colony hybridization screening of a partial genomic library with an ElPKS2 PCR product. ElPKS2 had a 6465-bp open reading frame that encoded 2155 amino acids and had introns of 56, 67, 54, and 71 bp. Although sequence analysis of the derived protein of ElPKS2 confirmed the polyketide synthase nature of its protein product, the function of that product remains unclear.

Wangiella (Exophiala) dermatitidis WdChs5p, a class V chitin synthase, is essential for sustained cell growth at temperature of infection.

The chitin synthase structural gene WdCHS5 was isolated from the black fungal pathogen of humans Wangiella dermatitidis. Sequence analysis revealed that the gene has a myosin motor-like-encoding region at its 5' end and a chitin synthase (class V)-encoding region at its 3' end. Northern blotting showed that WdCHS5 is expressed at high levels under conditions of stress. Analysis of the 5' upstream region of WdCHS5 fused to a reporter gene indicated that one or more of the potential regulatory elements present may have contributed to the high expression levels. Disruption of WdCHS5 produced mutants that grow normally at 25 degrees C but have severe growth and cellular abnormalities at 37 degrees C. Osmotic stabilizers, such as sorbitol and sucrose, rescued the wild-type phenotype, which indicated that the loss of WdChs5p causes cell wall integrity defects. Animal survival tests with a mouse model of acute infection showed that all wdchs5Delta mutants are less virulent than the parental strain. Reintroduction of the WdCHS5 gene into the wdchs5Delta mutants abolished the temperature-sensitive phenotype and reestablished their virulence. We conclude that the product of WdCHS5 is required for the sustained growth of W. dermatitidis at 37 degrees C and is of critical importance to its virulence.