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 (wdtup1Δ) 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 wdtup1Δ mutants, unlike those of the wild type, were arrested at a stage prior to the formation of true hyphae and subsequent conidia production.
morphogenesis; gene disruption; pseudohyphae; moniliform hyphae
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°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.
pH; transcription factor; Na+ stress; filamentous growth; conidiation; morphogenesis
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°C and consequently virulence. This report describes the expression in E. coli of a 12 kDa 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.
class V chitin synthase; black fungus; enzyme immunocytolocalization; phaeohyphomycosis agent
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.
Immunoaffinity purification; Transmembrane protein; Chitin synthase; Black pathogenic fungus
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.
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°C and were as virulent as the wild type in a mouse model. However, detailed morphological and cytological studies of the wdchs1Δ 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 wdchs2Δ 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°C and was not viable at 37°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.
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°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°C. Ectopic overexpression of WdSTUA repressed the convoluted colony surface growth on YPDA at 37°C, and also strongly repressed hyphal growth on PDA at 25°C and 37°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.
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.
We analyzed the effects of disrupting WdPKS1 on dihydroxynaphthalene melanin production and resistance to antifungal compounds. Transmission electron microscopy revealed that wdpks1Δ-1 yeast had thinner cell walls that lacked an electron-opaque layer compared to wild-type cells. However, digestion of the wdpks1Δ-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 wdpks1Δ-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.
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.
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°C but have severe growth and cellular abnormalities at 37°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 wdchs5Δ mutants are less virulent than the parental strain. Reintroduction of the WdCHS5 gene into the wdchs5Δ 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°C and is of critical importance to its virulence.
The chitin synthase structural gene WdCHS2 was isolated
by screening a subgenomic DNA library of Wangiella
dermatitidis by using a 0.6-kb PCR product of the gene as a
probe. The nucleotide sequence revealed a 2,784-bp open reading frame,
which encoded 928 amino acids, with a 59-bp intron near its 5′ end.
Derived protein sequences showed highest amino acid identities with
those derived from the CiCHS1 gene of Coccidioides
immitis and the AnCHSC gene of Aspergillus
nidulans. The derived sequence also indicated that WdChs2p is an
orthologous enzyme of Chs1p of Saccharomyces cerevisiae,
which defines the class I chitin synthases. Disruptions of
WdCHS2 produced strains that showed no obvious
morphological defects in yeast vegetative growth or in ability to carry
out polymorphic transitions from yeast cells to hyphae or to isotropic
forms. However, assays showed that membranes of wdchs2Δ
mutants were drastically reduced in chitin synthase activity. Other
assays of membranes from a
mutant showed that their residual chitin synthase activity was
extremely sensitive to trypsin activation and was responsible for the
majority of zymogenic activity. Although no loss of virulence was
detected when wdchs2Δ strains were tested in a mouse
model of acute infection, wdchs2Δwdchs3Δ
disruptants were considerably less virulent in the same model, even
though wdchs3Δ strains also had previously shown no loss
of virulence. This virulence attenuation in the
wdchs2Δwdchs3Δ mutants was similarly
documented in a limited fashion in more-sensitive
cyclophosphamide-induced immunocompromised mice. The importance of
WdChs2p and WdChs3p to the virulence of W. dermatitidis was
then confirmed by reconstituting virulence in the double mutant by the
reintroduction of either WdCHS2 or WdCHS3 into
the wdchs2Δwdchs3Δ mutant background.
1,8-Dihydroxynaphthalene (1,8-DHN) is a fungal polyketide that contributes to virulence when polymerized to 1,8-DHN melanin in the cell walls of Wangiella dermatitidis, an agent of phaeohyphomycosis in humans. To begin a genetic analysis of the initial synthetic steps leading to 1,8-DHN melanin biosynthesis, a 772-bp PCR product was amplified from genomic DNA using primers based on conserved regions of fungal polyketide synthases (Pks) known to produce the first cyclized 1,8-DHN-melanin pathway intermediate, 1,3,6,8-tetrahydroxynaphthalene. The cloned PCR product was then used as a targeting sequence to disrupt the putative polyketide synthase gene, WdPKS1, in W. dermatitidis. The resulting wdpks1Δ disruptants showed no morphological defects other than an albino phenotype and grew at the same rate as their black wild-type parent. Using a marker rescue approach, the intact WdPKS1 gene was then successfully recovered from two plasmids. The WdPKS1 gene was also isolated independently by complementation of the mel3 mutation in an albino mutant of W. dermatitidis using a cosmid library. Sequence analysis substantiated that WdPKS1 encoded a putative polyketide synthase (WdPks1p) in a single open reading frame consisting of three exons separated by two short introns. This conclusion was supported by the identification of highly conserved Pks domains for a β-ketoacyl synthase, an acetyl-malonyl transferase, two acyl carrier proteins, and a thioesterase in the deduced amino acid sequence. Studies using a neutrophil killing assay and a mouse acute-infection model confirmed that all wdpks1Δ strains were less resistant to killing and less virulent, respectively, than their wild-type parent. Reconstitution of 1,8-DHN melanin biosynthesis in a wdpks1Δ strain reestablished its resistance to killing by neutrophils and its ability to cause fatal mouse infections.
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 wdcdc42G14V 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 wdcdc42G14V 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 wdcdc42T19N and wdcdc42D120A 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.
Class III chitin synthases are important for hyphal growth in some
filamentous fungi but are not found in yeasts. Using a specific PCR
product that encodes a portion of the class III chitin synthase of
W. dermatitidis as a probe, we isolated the chitin synthase
gene, WdCHS3, from this polymorphic melanized pathogen of
humans. Northern blotting showed that WdCHS3 was highly
expressed under stress conditions, such as the shift of cells to
temperatures commensurate with infection, or to conditions that induce
cellular morphogenesis in this fungus. Analysis of the 5′ upstream
sequence of WdCHS3 provided evidence for a negative
regulatory element at between −780 and −1600 bp. Western blotting
indicated that the production of the WdChs3p was temperature dependent
and temporally regulated. Disruption of WdCHS3 in a
wild-type strain and in two temperature-sensitive morphological mutants
resulted in significantly reduced chitin synthase activities but did
not obviously affect their morphologies, growth rates, chitin contents,
or virulence. This paradox suggested that the contributions of the high
levels of WdCHS3 gene expression and WdChs3p production in
strains subjected to stress reside in unknown or unexamined parts of
the life cycle of this ecologically poorly known member of the Fungi
Imperfecti. Nonetheless, this report presents the first evidence that
transcription of a chitin synthase gene is regulated by a negative
regulatory element in its 5′ upstream sequence.
By using improved transformation methods for Wangiella
dermatitidis, and a cloned fragment of its chitin synthase 4
structural gene (WdCHS4) as a marking sequence, the
full-length gene was rescued from the genome of this human pathogenic
fungus. The encoded chitin synthase product (WdChs4p) showed high
homology with Chs3p of Saccharomyces cerevisiae and other
class IV chitin synthases, and Northern blotting showed that
WdCHS4 was expressed at constitutive levels under all
conditions tested. Reduced chitin content, abnormal yeast clumpiness
and budding kinetics, and increased melanin secretion resulted from the
disruption of WdCHS4 suggesting that WdChs4p influences
cell wall structure, cellular reproduction, and melanin deposition,
respectively. However, no significant loss of virulence was detected
when the wdchs4Δ strain was tested in an acute mouse
model. Using a wdchs1Δ wdchs2Δ wdchs3Δ triple mutant
of W. dermatitidis, which grew poorly but adequately at
25°C, we assayed WdChs4p activity in the absence of activities
contributed by its three other WdChs proteins. Maximal activity
required trypsin activation, suggesting a zymogenic nature. The
activity also had a pH optimum of 7.5, was most stimulated by
Mg2+, and was more inhibited by polyoxin D than by
nikkomycin Z. Although the WdChs4p activity had a broad temperature
optimum between 30 to 45°C in vitro, this activity alone did not
support the growth of the wdchs1Δ wdchs2Δ wdchs3Δ
triple mutant at 37°C, a temperature commensurate with infection.
The occurrence of ferrichrome-type hydroxamate siderophores in soil was confirmed. In the presence of the iron-scavenging chelator ethylenediamine[di(o-hydroxyphenylacetic)acid], soil extract stimulated the growth of an Escherichia coli strain possessing the ferrichrome transport protein (TonA) but did not stimulate growth of a strain lacking this protein (TonA−). The siderophore concentration in a 1:1 (soil-water) extract was estimated to be approximately 78 nM. Specificity of the assay was supported by the absence of significant differential strain responses to ferric citrate, ferric 2,3-dihydroxybenzoate, enterochelin, ferrioxamine B, coprogen, and triacetylfusigen.
Yeast forms of the dimorphic fungus Cladosporium werneckii grow by polar budding and yield a homogeneous yeast phase when cultured at 21 C in an agitated sucrose-salts medium (Czapek-Dox broth). Yeast extract enrichment of such a yeast phase consisting of 104 yeasts per ml induces a quantitative conversion of the yeasts to true hyphae. This conversion is not mediated by a transition cell and is often attended by capsule formation. When 105 or 106 yeasts per ml receive enrichment, a nonquantitative conversion to moniliform hyphae is effected and no capsule formation is observed. Rapid agitation compared to slow agitation or stationary incubation of the nutritionally mediated conversion cultures greatly accelerates the production of lateral hyphal buds or their yeast progenies. These cells appear incapable of undergoing nutritional conversion to hyphae, but instead must grow for several generations in the unenriched sucrose-salts medium to restore conversion competence. Temperature shifts affect directly the morphology and morphogenesis of the yeast in unenriched medium; at 17 C yeasts are smaller and more ovoid than at 21 C, and at 30 C marked conversion of yeasts to moniliform hyphae occurs. A methodology employing the Coulter counter and Coulter channelizer provides evidence that direct correlations do not always exist between the optimum conditions for the growth of C. werneckii and the optimum conditions for its yeast-to-mold conversion.
Ultrastructural comparisons of yeast and hyphal bud formation in Phialophora dermatitidis reveal that bud initiation is characterized by a blastic rupture of the outer portion of the yeast or hyphal wall and the emergence of a bud protuberance through the resulting opening. The wall of the emerging bud is continuous, with only an inner wall layer of the parental yeast or hypha. The outer, ruptured portion of the parental wall typically forms a collar around the constricted emergence region of the developing bud. The cytoplasm within the very young emerging bud invariably contains a small number of membrane-bound vesicles. The septum formed between the daughter bud and the parental yeast or hypha is a complete septum devoid of a septal pore, septal pore plug, or any associated Woronin bodies characteristic of simple septa of the moniliform or true hyphae. These observations suggest that yeast bud formation and lateral hyphal bud formation in the dimorphic fungus P. dermatitidis involve a growth process which occurs identically in both the yeast and mold phase of this human pathogenic organism.
Phase-contrast microscope observations of the dimorphic fungus Phialophora dermatitidis revealed that thick-walled yeasts often tended to form aggregates and then to conjugate. Fusions were also observed among hyphae derived from the thick-walled yeasts.
The details of the morphological and structural events occurring during yeast-to-mold conversion of the human pathogenic fungus Phialophora dermatitidis as seen by phase-contrast microscopy and electron microscopy are described and illustrated. Budding yeasts growing exponentially were observed to have thin walls and a cytoplasm exhibiting the characteristics of rapidly growing cells including numerous mitochondria, abundant ribosomes, few vacuoles, and little accumulation of storage material. In contrast, thick-walled yeasts were characterized by less apparent or significantly fewer mitochondria and ribosomes and the presence of considerable amounts of storage materials. Microscope observations of yeast-to-mold conversion revealed that only thick-walled yeasts having prominent lipid bodies in their cytoplasm converted to hyphal forms. Typically, the thick-walled yeast formed two to a number of moniliform hyphal cells which in turn often produced true hyphae. The results indicated that yeasts of P. dermatitidis must acquire spore-like characteristics by becoming thick-walled and by accumulating considerable endogenous substrate reserves before they convert and produce hyphae.
Hyphal wall compositions of six Leptosphaeria species were compared to assess whether gross changes have occurred in the hyphal wall chemistry of closely related fungi which have become ecologically restricted to marine or terrestrial habitats. Unfractionated, lipid-extracted hyphal walls of each Leptosphaeria species had qualitatively identical compositions consisting of glucose, mannose, galactose, glucosamine, amino acids, and traces of galactosamine. Quantitative analyses showed that the hyphal wall components varied from species to species. Qualitative compositions of alkali-soluble wall fractions from each species were identical and contained the same sugars found in the unfractionated walls. The alkali-insoluble residues contained glucose, glucosamine, and amino acids. The alkali-soluble fractions were composed predominantly of glucose, galactose, and mannose. The alkali-insoluble fractions contained high concentrations of glucose and glucosamine and relatively low concentrations of amino acids.
The spent seawater medium of 4-day-old-cultures of the filamentous marine fungus Leptosphaeria albopunctata had a high viscosity after the fungus was collected by high-speed centrifugation. Microscopic examination of uncentrifuged mycelium suspended in India ink revealed that the viscosity resulted from capsular material. These capsules became disassociated from the mycelium during centrifugation. Precipitation of the medium of centrifuged cultures with 95% ethyl alcohol yielded a highly anthrone-positive polysaccharide material, composed of large amounts of glucose and minute amounts of mannose. Time course studies of the nutritional requirements for capsular polysaccharide production revealed that the capsular material was produced in large amounts, and on a wide variety of sugars, during the period of rapid growth, but was quickly degraded and presumably remetabolized in older cultures. The amount of capsular material produced was enhanced by NaCl concentrations above that of artificial seawater, and KCl could be substituted for NaCl. The salts MgCl2 and CaCl2 were also required for capsule production by L. albopunctata, although growth was obtained in cultures without added amounts of these constituents. The possible role of these salts in the metabolism of the fungus is discussed.
Hydrolytic residues of the cell walls of 48 strains of Actinoplanaceae, previously assigned to 10 species and the four genera, Actinoplanes, Ampullariella, Amorphosporangium, and Pilimelia, were examined by paper chromatography and column chromatography. Comparisons were made for taxonomic purposes between the groupings obtained, by use of chemical characters and the groupings currently recognized morphologically. Most of the species investigated had qualitatively distinct cell wall compositions. Often, however, the cell wall compositions of species in different genera were more similar, in some respects, than were those of species in the same genus. Quantification of the cell wall amino acids and amino sugars substantiated that cross-generic similarities existed. Based on these results and the morphological conclusions reached by other investigators, a single-genus concept is suggested for the Actinoplanaceae examined.