Comparative genomics of opportunistic Phialophora species involved in divergent disease types.

BACKGROUND: Black opportunists Phialophora verrucosa complex species can cause different disease types in competent and in immunocompromised individuals, but are remarkably overrepresented in CARD9-related infections. OBJECTIVES: To better understand the ecology and potential pathogenicity of opportunistic Phialophora species and reveal eventual genetic parameters associated with the behaviour in vivo and genetic profiles in patients with CARD9 immunodeficiency. METHODS: Genomes of 26 strains belonging to six species of the Phialophora verrucosa complex were sequenced. Using multilocus analysis, all environmental and clinical strains were identified correctly. We compared the genomes of agents from different disease types among each other including CARD9 immunodeficiency. RESULTS: We obtained genome sizes of the 26 Phialophora strains ranged between 32 and 37 MB. Some species showed considerable intraspecific genomic variation. P americana showed the highest degree of variability. P verrucosa was variable in CAZy enzymes, whereas P americana varied in PKS-related genes. Phialophora species, particularly P verrucosa, are relatively frequent in patients with CARD9-related immunodeficiency. Different mutations in the CARD9 gene seem to increase susceptibility for infection by different groups of species, that is either Candida, dermatophytes or black fungi. A number of patients with chromoblastomycosis revealed an as yet unknown CARD9 mutation. TNFα impairment was prevalent in patients with CARD9 infections, while CBM patients were invariably IFNγ. CONCLUSIONS: From genomic investigations, the known virulence factors between clinical and environmental strains did not reveal any significant difference. Phialophora complex has an equal chance to cause infection in humans, either healthy or CARD9-impaired.

Biochemical Characterization and Mutational Analysis of a Lactone Hydrolase from Phialophora americana.

The mycotoxin zearalenone (ZEN) is a secondary metabolite produced mainly by Fusarium species. ZEN poses health hazards both for humans and animals, as a major contaminant in the food and feed industries. Currently, there is no effective technique for degrading ZEN during industrial processes. In this study, we isolated and biochemically characterized a novel lactone hydrolase, ZHD607, isolated from Phialophora americana, cloned, and exogenously expressed in Pichia pastoris. ZHD607 was characterized as a mesophilic lactone hydrolase having a neutral pH and showing optimal activity at 35 °C and pH 8.0. Two mutants, ZHDM1 and I160Y, generated from ZHD607 based on structure and sequence alignment analyses, exhibited 2.9- and 3.4-fold higher activity towards ZEN than did ZHD607. Molecular dynamics simulation revealed diverse mechanisms driving this improved catalytic activity. These findings enrich our knowledge about ZHD enzyme family and represent an important step toward industrialization of ZEN-detoxifying lactone hydrolases.

Phialophora section Catenulatae disassembled: New genera, species, and combinations and a new family encompassing taxa with cleistothecial ascomata and phialidic asexual states.

The Pleuroascaceae (Leotiomycetes) is introduced for Phialophora hyalina (section Catenulatae) and its closest relatives based on analyses of DNA sequences of five gene regions and the comparison of cultural and micromorphological characters. The family is resolved as a strongly supported clade that encompasses Pleuroascus and the new anamorph genera Entimomentora and Venustampulla. The latter includes V. parva, a species placed formerly in Scopulariopsis, and V. echinocandica, which is established for the echinocandin-producing isolate BP-5553. Entimomentora includes E. hyalina, a species based on the ex-type strain of Ph. hyalina. Additional isolates identified as Ph. hyalina are distantly related to the Pleuroacaceae and include Psychrophila antarctica (Arachnopezizaceae) and Cryonesomyces dreyfussii, the sole member of the new genus Cryonesomyces (incertae sedis). Isolates identified or deposited as Ph. alba are also not closely related; they include a species for which we propose the name Neobulgaria koningiana (Gelatinodiscaceae) and a second psychrophilic species that we describe as Psychrophila lagodekhiensis. Of the 13 isolates assessed for in vitro antifungal activity, only V. echinocandica inhibited the growth of Candida albicans.

The genus Anthopsis and its phylogenetic position in Chaetothyriales.

The genus Anthopsis was introduced for a black fungus with peculiar, inverted phialides and triangular conidia. The genus accommodates, in addition to the type species Anthopsis deltoidea, which once was reported as a cause of human phaeohyphomycosis, two further taxa: A. catenata and A. microspora. Current taxonomy is mainly based on microscopic structures of phialides. To assess the phylogenetic position of the genus, sequences of the internal transcribed spacer region and partial LSU rDNA were obtained for Anthopsis spp. and compared with sequences from public databases. Phylogenetic analyses based on both loci were used to assess the evolutionary relationships of Anthopsis spp. at the family and ordinal levels. Anthopsis s.str. was found to cluster in Chaetothyriales, while A. catenata proved to be of helotialean affinity. Thermotolerance and morphology of each species were recorded.

Identification and typing of isolates of Cyphellophora and relatives by use of amplified fragment length polymorphism and rolling circle amplification.

The species diversity and identification of black fungi belonging to Cyphellophora and Phialophora, which colonize and infect human skin and nails, were studied using amplified fragment length polymorphism (AFLP). A total of 76 Cyphellophora and Phialophora isolates were evaluated, and their delimitation was compared to earlier studies using multilocus sequencing. The results of the AFLP analysis and sequencing were in complete agreement with each other. Seven species-specific padlock probes for the most prevalent species were designed on the basis of the ribosomal DNA internal transcribed spacer region, and identification of the respective species could easily be achieved with the aid of rolling circle amplification.

Recalcitrant primary subcutaneous phaeohyphomycosis due to Phialophora verrucosa.

Phialophora verrucosa has rarely been reported for causing phaeohyphomycosis, which tends to occur in immunocompromised individuals. The case of primary subcutaneous phaeohyphomycosis due to P. verrucosa in an otherwise healthy Chinese female is presented. The girl presented with asymptomatic skin lesions when she was only 16 year old. Histological examinations revealed multiple dematiceous hyphael elements in the dermis and subcutaneous tissues. Fungal cultures were identified as P. verrucosa repeatedly based on the morphological features and confirmed by the internal transcribed spacer region nucleotide sequencing. The infection was so extremely recalcitrant that prolonged systemic antifungal regimens for 12 years revealed limited effect. The skin lesions slowly progressed and caused marked disfigurement despite the encouraging results of in vitro susceptibility. However, no relevant side effects have been reported in the course, and the patient gave birth to a healthy baby while under the maintenance treatment of itraconazole and terbinafine. This case is special in terms of the early onset, the rare clinical aspect of the pathogen, the discrepancy between in vitro and in vivo antifungal activities and especially the prolonged and recalcitrant course in such an otherwise healthy host.

Purification, gene cloning and characterization of an acidic ß-1,4-glucanase from Phialophora sp. G5 with potential applications in the brewing and feed industries.

An extracellular ß-1,4-glucanase (CelG5, ∼55.0 kDa) was isolated from the culture filtrate of Phialophora sp. G5, and its encoding gene was cloned. The deduced amino acid sequence of CelG5 was at most 73.6% and 44.0%, respectively, identical with a hypothetical protein from Sordaria macrospora and an experimentally verified GH 7 endo-ß-1,4-glucanase of Neurospora tetrasperma FGSC 2508. Native CelG5 had pH and temperature optima of pH 4.5-5.0 and 55-60°C. The enzyme showed some properties superior than most fungal ß-1,4-glucanases, such as high activity over a wide pH range (exhibiting >50% of the maximum activity at pH 2.0-7.0), excellent stability in extreme acidic to alkaline conditions (pH 2.0-9.0), and strong resistance against pepsin and trypsin (retaining 89% and 94% activity, respectively). Recombinant CelG5 produced in Pichia pastoris had a molecular mass and a pH optimum similar to native CelG5, but with maximal activity at 65°C. Application tests showed that native CelG5 was stable under simulated gastric conditions (retaining >70% activity), and had capacity to decrease the viscosity of barley-bean feed (8.9% by 200 U CelG5) and mash (6.1% by 50 U CelG5) and increase the filtration rate of mash (18.4% by 50 U CelG5). These properties make CelG5 a good candidate for utilization in the animal feed and brewing industries.

A thermophilic cellulase complex from Phialophora sp. G5 showing high capacity in cellulose hydrolysis.

A cellulase-producing mesophilic fungal strain, named G5, was isolated from the acidic wastewater and mud of a tin mine and identified as Phialophora sp. based on the internal transcribed spacer sequence. The volumetric activities and specific activities of cellulase induced by different carbon sources (Avicel, corn cob, wheat bran and corn stover) were compared. The cellulase complex of Phialophora sp. G5 exhibited the optimal activities at 60-65 °C and pH 4.0-5.0, and had good long-term thermostability at 50 °C. Compared with the commercial cellulase (Accellerase 1500, Genencor), the enzyme under study showed 60% and 80% of the capacity to hydrolyze pure cellulose and natural cellulose, respectively. This is the first study to report that a cellulytic enzymes complex from Phialophora genus, and the superior properties of this enzyme complex make strain G5 a potential microbial source to produce cellulase for industrial applications, and the production ability could be improved by mutagenesis.

Occurrence and characteristics of group 1 introns found at three different positions within the 28S ribosomal RNA gene of the dematiaceous Phialophora verrucosa: phylogenetic and secondary structural implications.

BACKGROUND: Group 1 introns (ribozymes) are among the most ancient and have the broadest phylogenetic distribution among the known self-splicing ribozymes. Fungi are known to be rich in rDNA group 1 introns. In the present study, five sequences of the 28S ribosomal RNA gene (rDNA) regions of pathogenic dematiaceous Phialophora verrucosa were analyzed using PCR by site-specific primers and were found to have three insertions, termed intron-F, G and H, at three positions of the gene. We investigated the distribution of group 1 introns in this fungus by surveying 34 strains of P. verrucosa and seven strains of Phialophora americana as the allied species. RESULTS: Intron-F's (inserted at L798 position) were found in 88% of P. verrucosa strains, while intron-G's (inserted at L1921) at 12% and intron-H's (inserted at L2563) at 18%. There was some correlation between intron distribution and geographic location. In addition, we confirmed that the three kinds of introns are group 1 introns from results of BLAST search, alignment analysis and Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). Prediction of secondary structures and phylogenetic analysis of intron sequences identified introns-F and G as belonging to subgroup IC1. In addition, intron-H was identified as IE. CONCLUSION: The three intron insertions and their insertion position in the 28S rDNA allowed the characterization of the clinical and environmental isolates of P. verrucosa and P. americana into five genotypes. All subgroups of introns-F and G and intron-H were characterized and observed for the first time in both species.

Real-time PCR assays for the quantification of Phialophora gregata f. sp. sojae IGS genotypes A and B.

Populations of Phialophora gregata f. sp. sojae, the causal agent of brown stem rot (BSR) of soybean, consist of two genotypes, designated A and B. These genotypes are differentiated by an insertion or deletion in the intergenic spacer region (IGS) of ribosomal DNA. The two genotypes differ in the type and severity of symptoms they cause and have displayed preferential host colonization. Methods to quantify populations of P. gregata f. sp. sojae and to distinguish between the two genotypes are essential to understanding this host-pathogen interaction and to improving control of BSR. A real-time, quantitative polymerase chain reaction (qPCR) assay was developed for the specific detection and quantification of P. gregata f. sp. sojae genotype A. This assay is specific to P. gregata f. sp. sojae genotype A, sensitive to 50 fg of DNA, and unaffected by the presence of soybean or soil DNA. When the P. gregata f. sp. sojae genotype A-specific primer/probe set is used in a multiplex qPCR assay with a previously developed primer/probe set which indiscriminately amplifies both genotypes, the quantity of P. gregata f. sp. sojae genotype B can be indirectly determined. This multiplex assay provides a rapid and robust method for studying both the population size and genetic structure of P. gregata f. sp. sojae in its soybean host and in the soil.

Colorimetric in situ hybridization (CISH) with digoxigenin-labeled oligonucleotide probes in autofluorescent hyphomycetes.

We used digoxigenin-labeled probes for in situ hybridization of hyphomycetes to replace the commonly used fluorescent proof of probe binding by a colorimetric reaction. The resulting blue-purple, intracellular precipitate could be easily detected by light microscopy, and thus presented a promising method to overcome autofluorescence of fungal material and substratum. Optimal cell fixation and permeabilization procedures, as well as hybridization conditions were developed on the example of two different hyphomycetes: Phialophora sp. and Cartapip, a colorless mutant of Ophiostoma piliferum (Agra Sol).

A new species, Phialophora europaea, causing superficial infections in humans.

A new species, Phialophora europaea, member of the P. verrucosa complex, is introduced. It is distinguished from existing species by reduced, flaring phialidic collarettes and inability to assimilate melibiose as sole source of carbon. Analysis of ITS1 and 2 rDNA of six strains attributed to the species show it to be clearly individualized. All strains originated from cutaneous and nail infections of humans in North-western Europe. A key to morphologically similar taxa is provided.

Mitochondrial DNA analysis of Phialophora verrucosa.

Restriction fragment length polymorphism (RFLP) of mitochondrial DNA (mtDNA) was examined in 32 isolates of Phialophora verrucosa (eight isolates from Japan, 10 from China, four from the USA, six from Venezuela and four from Colombia) and in three of Phialophora americana using five restriction enzymes. P. verrucosa isolates were divided into 10 mtDNA types based on RFLP patterns. Phylogeny constructed on sequence divergence of mtDNA indicated that P. verrucosa is a single species and isolates are clustered into three groups. Japan and the USA contained Group A and Group B isolates, China Group B isolates and South America Group B and Group C isolates. RFLP patterns of P. americana mtDNA were identical to those of Type 1 or Type 4 of P. verrucosa mtDNA, suggesting that both are identical. RFLP patterns of P. verrucosa were distinct from those of other dematiaceous fungi including Exophiala jeanselmei, E. moniliae, E. dermatitidis, E. spinifera, Cladophialophora (Cladosporium) carrionii, Fonsecaea pedrosoi, and Hortaea werneckii. These results indicate that RFLP analysis of mtDNA is a useful method for the identification, taxonomy, typing, epidemiology and phylogeny of P. verrucosa.

Use of the polymerase chain reaction to identify coding sequences for chitin synthase isozymes in Phialophora verrucosa.

Based on conserved amino-acid regions predicted for the chitin synthases (Chs) of Saccharomyces cerevisiae, two different primer sets were synthesized and used in polymerase chain reactions (PCRs) to amplify 614-bp and 366-bp sequences from genomic DNA of the zoopathogenic fungus Phialophora verrucosa. DNA-sequencing and Southern-blotting analyses of the 614-bp DNA amplification products suggested that portions of two distinct P. verrucosa chitin synthase genes (PvCHS1, PvCHS2), coding for two different zymogenic-type PvChs isozymes, had been identified. The deduced amino-acid sequence of each fell into different Chs classes, namely class I and class II. In addition, the 366-bp DNA segment was shown to code for a conserved region having homology with the CSD2/CAL1 gene of S. cerevisiae, which encodes a nonzymogenic-type enzyme, Chs3, in that fungus. The amino-acid sequence derived from PvCHS3 exhibits 88.2% similarity and 78.4% identity to the same amino-acid region of the S. cerevisiae enzyme. These results provide a critical first step toward investigating the molecular and pathogenic importance of CHS gene regulation in this fungus and for exploring steps leading to Chs function as potential targets for the design of new therapeutic agents.

Nuclear division in temperature-sensitive multicellular mutants of Wangiella dermatitidis.

Temperature-sensitive morphological mutants of Wangiella dermatitidis were stained with mithramycin, a fluorescent nuclear stain. Multicellular forms of the mutants each contained two or more nuclei, documenting that nuclear division accompanied the yeast-to-multicellular form conversion.