New Δ8,9-pregnene steroids isolated from the extremophile fungus Exophiala oligosperma.

Three pregnene steroids, two of them new, were isolated from ethyl-acetate partition of liquid-cultivation of the extremophyle fungus Exophiala oligosperma, found in a pH 1.5 hydrochloric acid aqueous solution. Spectroscopic studies using NMR and HRMS, allowed the identification of the molecular structures of both Δ8,9-pregnenes, still not described in the literature.

Exophiarin, an isocoumarin from the fungus Exophiala sp. with antihyperglycemic activity.

Chemical characterization of ethyl acetate extract of Exophiala sp. has afforded the isolation of three compounds including a new isocoumarin named exophiarin (1). Exophiala sp. was obtained from the soil containing dumped organic waste (litter). Initially, LC-UV-MS analysis of the extract of Exophiala sp. revealed the presence of a new compound having molecular weight 438 (1) and previously reported TPI-2 and TPI-5. The novelty was established using advanced database search comprising of biological source, molecular weight and UV profile. 1D, 2D NMR and HRMS data have been used for structure elucidation. Exophiarin with TPI-2 and TPI-5 have displayed moderate improvement in glucose uptake activity when tested in rat skeletal muscle cell line L6.

[A comparison of biosurfactant production between clinical and environmental Exophiala isolates]. / Klinik ve çevresel Exophiala izolatlarinda biyosurfaktan üretiminin karsilastirilmasi.

Black yeast in the genus Exophiala are able to grow in hydrocarbon-contaminated environments and are pathogenic in immunosuppressed hosts. The biosurfactant produced by Exophiala species may be associated with strain pathogenicity by changing the hydrophobicity. The aim of this study was to prove the hypothesis that biosurfactant production in Exophilia strains isolated from clinical samples is lower than the strains isolates from toxic (dishwasher and railway sleepers) environments. A total of 122 Exophiala isolates 108 environmental (isolated from 82 dishwashers and 36 railway sleepers) and 14 clinical isolates confirmed by molecular tests were included in the study. Biosurfactant activity was tested by the drop collapse method, in which the surface of a microtiter plate well was evaluated for the presence of a biosurfactant, and by the oil spreading technique on crude oil. An open source analyses program, ImageJ®, was used for crude oil spreading technique data. A clear surface zone that differs more than two standard deviations from the mean size was accepted as a positive result. Among the 122 Exophiala species, 11 (9.0%) and 10 (8.2%) strains showed biosurfactant activity by the drop collapse test and oil spreading method, respectively. An acceptable relation was found between the drop collapse test and oil spreading method (Cohen κ coefficient= 0.30). Despite the presence of isolates showing biosurfactant activity, no statistically significant difference was detected between Exophiala strains (p= 0.72). The biosurfactant levels of environmental isolates were higher than the isolates obtained from the patients (p= 0.03). The highest biosurfactant level was observed in one Exophiala phaeomuriformis strain isolated from a dishwasher. There was no difference between the biosurfactant levels of the dishwasher and railway sleeper isolates (p= 0.66). Biosurfactant production may be a more important determinant of virulence in Exophiala species than expected. In this study, biosurfactant activity was higher in environmental isolates compared to the clinical isolates. Consensus of multiple biosurfactant screening protocols may clarify why environmental Exophiala species are less virulent. Further studies should evaluate biosurfactant activity in additional clinical Exophiala isolates. The biosurfactant activity of more Exophiala isolates obtained from patients should be investigated with further planned studies.

Exploration of insecticidal potential of an alpha glucosidase enzyme inhibitor from an endophytic Exophiala spinifera.

AIM: The present study aimed to isolate and screen endophytes from Trachyspermum ammi with the ability to inhibit alpha glucosidase enzyme and evaluate their insecticidal potential. METHODS AND RESULTS: Endophytic fungi isolated from T. ammi were screened for alpha glucosidase inhibitory activity. Maximum inhibition (96%) was observed in an isolate AZ-9, identified to be Exophiala spinifera on morphological and molecular basis. Production of fungal metabolites was carried out in malt extract broth followed by extraction with ethyl acetate. Brown coloured gummy residue obtained after evaporation of ethyl acetate was partially soluble in water yielding white precipitates. The precipitate exhibiting α-glucosidase inhibitory activity was purified by repeated washing and centrifugation. The insecticidal activity of inhibitor was evaluated on Spodoptera litura (Fab.) by feeding this pest on diet amended with inhibitor. It resulted in significant larval mortality as well as deformities in emerging adults. A reduction in vivo digestive enzyme activity was also observed. Nutritional analysis revealed the toxic effect of AZ-9 inhibitor on various food utilization parameters of S. litura. A significant reduction was recorded in relative growth and consumption rate of S. litura. CONCLUSIONS: This is the first report on production of an alpha glucosidase inhibitor from E. spinifera with insecticidal activity. SIGNIFICANCE AND IMPACT OF THE STUDY: The study highlights the importance of endophytes in providing protection against insect pests to the host. It also suggests the insecticidal potential of alpha glucosidase inhibitor from E. spinifera against polyphagous pest S. litura.

Rapid Identification of Clinically Relevant Members of the Genus Exophiala by Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry and Description of Two Novel Species, Exophiala campbellii and Exophiala lavatrina.

Exophiala is a ubiquitous pleomorphic genus comprising at least 40 species, many of which have been associated with superficial, visceral, or systemic infections in humans, other mammals, or cold-blooded animals. In this study, we investigated the potential of matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF MS) for the identification of Exophiala species. A total of 89 isolates (including 50 human and 4 animal clinical isolates) stored in the National Collection of Pathogenic Fungi were identified by PCR amplification and sequencing of internal transcribed spacer region 1. Eighty-three of the isolates corresponded to 16 known species within Exophiala/Rhinocladiella The remaining six isolates are shown by phylogenetic analyses based on four loci to represent two novel Exophiala species. Four isolates from domestic bathrooms which form a sister species with Exophiala lecanii-corni are described here as Exophiala lavatrina sp. nov. The remaining two isolates, both from subcutaneous infections, are distantly related to Exophiala oligosperma and are described here as Exophiala campbellii sp. nov. The triazoles and terbinafine exhibited low MICs against all Exophiala isolates in vitro MALDI-TOF MS successfully distinguished all 18 species and identified all isolates after appropriate reference spectra were created and added to commercial databases. Intraspecific mean log scores ranged from 1.786 to 2.584 and were consistently significantly higher than interspecific scores (1.193 to 1.624), with the exception of E. lecanii-corni and E. lavatrina, for which there was considerable log score overlap. In summary, MALDI-TOF MS allows the rapid and accurate identification of a wide range of clinically relevant Exophiala species.

ATR-FTIR Spectroscopy Highlights the Problem of Distinguishing Between Exophiala dermatitidis and E. phaeomuriformis Using MALDI-TOF MS.

The present study compared two chemical-based methods, namely, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy, to understand the misidentification of Exophiala dermatitidis and Exophiala phaeomuriformis. The study utilized 44 E. dermatitidis and 26 E. phaeomuriformis strains, which were partially treated with strong acids and bases for further evaluation. MALDI-TOF MS and ATR-FTIR spectroscopy data of the two Exophiala species were compared. Data groupings were observed for the chromic acid- and nitric acid-treated species when the black yeast sources were categorized as creosoted-oak sleepers, concrete sleepers, or dishwasher isolates. The MALDI-TOF MS data for the metalloenzyme-containing regions were consistent with the ATR-FTIR spectroscopy data. These results indicated that environmental isolates might contain metals not found in human isolates and might interfere with chemical-based identification methods. Therefore, MALDI-TOF MS reference libraries should be created for clinical strains and should exclude petroleum-associated environmental isolates.

Diversity of exophillic acid derivatives in strains of an endophytic Exophiala sp.

Members of the fungal genus Exophiala are common saprobes in soil and water environments, opportunistic pathogens of animals, or endophytes in plant roots. Their ecological versatility could imply a capacity to produce diverse secondary metabolites, but only a few studies have aimed at characterizing their chemical profiles. Here, we assessed the secondary metabolites produced by five Exophiala sp. strains of a particular phylotype, isolated from roots of Microthlaspi perfoliatum growing in different European localities. Exophillic acid and two previously undescribed compounds were isolated from these strains, and their structures were elucidated by spectroscopic methods using MS, 1D and 2D NMR. Bioassays revealed a weak activity of these compounds against disease-causing protozoa and mammalian cells. In addition, 18 related structures were identified by UPLC/MS based on comparisons with the isolated structures. Three Exophiala strains produced derivatives containing a ß-d-glucopyranoside moiety, and their colony morphology was distinct from the other two strains, which produced derivatives lacking ß-d-glucopyranoside. Whether the chemical/morphological strain types represent variants of the same genotype or independent genetic populations within Exophiala remains to be evaluated.

MALDI-TOF MS-based identification of black yeasts of the genus Exophiala.

In this study, we investigated the applicability of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the identification of Exophiala species. The analysis included a total of 110 Exophiala isolates, including 15 CBS strains representing 4 species, Exophiala dermatitidis (61), E. phaeomuriformis (36), E. crusticola (9), and E. heteromorpha (4), that had been previously identified based on internal transcribed spacer (ITS) regions. We also compared the relative efficacies of Sabouraud glucose agar (SGA) and Columbia agar (CA) for use in MALDI-TOF MS. Remarkably, we obtained a log-score value ≥2.0 by using either SGA or CA for all 15 CBS strains, indicating species-level identification. The remaining 95 Exophiala strains were identified to the genus or species levels, with identification rates of 96.8% and 90.5%, using SGA or CA, respectively. Most of the E. dermatitidis (100% and 92.9%), E. phaeomuriformis (80.6% and 83.9%), E. crusticola (50% and 100%), and E. heteromorpha (100% and 100%) isolates were correctly identified using SGA or CA, respectively. Furthermore, 58.9% and 26.3% of the strains had log-score values of ≥2.0 by using SGA and CA, respectively. Our results indicate that MALDI-TOF MS is a rapid and reliable technique with high rates of correct taxonomic identification.

Analyses of black fungi by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS): species-level identification of clinical isolates of Exophiala dermatitidis.

Conventional mycological identifications based on the recognition of morphological characteristics can be problematic. A relatively new methodology applicable for the identification of microorganisms is based on the exploitation of taxon- specific mass patterns recorded from abundant cell proteins directly from whole-cell preparations, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). This study reports the application of MALDI-TOF MS for the differentiation and identifications of black yeasts, isolated from the respiratory tracts of patients with cystic fibrosis (CF). Initial phenotypic and DNA sequence-based analyses identified these isolates to be Exophiala dermatitidis. The type strains of E. dermatitidis (CBS 207.35(T)) and other species of Exophiala were included in the MALDI-TOF MS analyses to establish the references for comparing the mass spectra of the clinical isolates of Exophiala. MALDI-TOF MS analyses exhibited extremely close relationships among the clinical isolates and with the spectra generated from the type strain of E. dermatitidis. The relationships observed between the E. dermatitidis strains from the MALDI-TOF MS profiling analyses were supported by DNA sequence-based analyses of the rRNA ITS1 and ITS2 regions. These data demonstrated the applicability of MALDI-TOF MS as a reliable, rapid and cost-effective method for the identification of isolates of E. dermatitidis and other clinically relevant fungi and yeasts that typically are difficult to identify by conventional methods.

Use of styrene as sole carbon source by the fungus Exophiala oligosperma: optimization and modeling of biodegradation, pathway elucidation, and cell membrane composition.

Biodegradation of styrene by Exophiala sp. was tested at different initial concentrations (19.3-170.6 mgl(-1)), pH (2.8-8.7), and temperatures (19.8-45.1 °C), for 120 h according to a 2(3) full-factorial central composite design. The specific growth rate (SGR, per hour) and specific styrene utilization rate (SUR, milligrams of styrene per milligram of biomass per hour) values were used as the response variables for optimization purposes. The interactions between concentration and temperature (P=0.022), and pH and temperature (P=0.010) for SGR, and interactions between concentration and temperature (P=0.012) for SUR were found to be statistically significant. The optimal values for achieving high SGR (0.15 h(-1)) and SUR (0.3622 mg styrene mg(-1) biomass h(-1)) were calculated from the regression model equation. Those values are C(o)=89.1 mgl(-1), pH=5.4, and T=31.5 °C for SGR and C(o)=69.2 mgl(-1), pH=5.5, and T=32.4 °C for SUR. It was also observed that the Exophiala strain degrades styrene via phenylacetic acid, involving initial oxidation of the vinyl side chain. Besides, in the presence of styrene, changes in the fatty acids profile were also observed. It is hypothesized that an increasing amount of linoleic acid (18:2) may be involved in the protection of the fungus against toxic substrate.

Fungal melanins differ in planar stacking distances.

Melanins are notoriously difficult to study because they are amorphous, insoluble and often associated with other biological materials. Consequently, there is a dearth of structural techniques to study this enigmatic pigment. Current models of melanin structure envision the stacking of planar structures. X ray diffraction has historically been used to deduce stacking parameters. In this study we used X ray diffraction to analyze melanins derived from Cryptococcus neoformans, Aspergillus niger, Wangiella dermatitides and Coprinus comatus. Analysis of melanin in melanized C. neoformans encapsulated cells was precluded by the fortuitous finding that the capsular polysaccharide had a diffraction spectrum that was similar to that of isolated melanin. The capsular polysaccharide spectrum was dominated by a broad non-Bragg feature consistent with origin from a repeating structural motif that may arise from inter-molecular interactions and/or possibly gel organization. Hence, we isolated melanin from each fungal species and compared diffraction parameters. The results show that the inferred stacking distances of fungal melanins differ from that reported for synthetic melanin and neuromelanin, occupying intermediate position between these other melanins. These results suggest that all melanins have a fundamental diffracting unit composed of planar graphitic assemblies that can differ in stacking distance. The stacking peak appears to be a distinguishing universal feature of melanins that may be of use in characterizing these enigmatic pigments.

Sample preparation and 2-DE procedure for protein expression profiling of black microcolonial fungi.

The ecology and stress adaptation of black rock inhabiting fungi in hot and cold extreme environments are not yet well understood. Two-dimensional gel electrophoresis (2-DE) is a promising tool to study the protein expression profiling and the metabolic status of microorganisms under stress conditions. The sample preparation has been shown to be the bottleneck for high resolution protein separation in 2-DE. For this purpose conditions must be optimized to obtain reliable and reproducible results. In addition, due to a multilayered and strongly melanized cell wall of black microcolonial fungi, special protocols for cell disruption and processing are required. In the present study, the protocol for protein extraction was established and optimized for the black yeast Exophiala jeanselmei MA 2853. The same protocol was successfully examined also for the meristematic fungus Coniosporium perforans MA 1299. Among the three procedures evaluated, trichloroacetic acid (TCA) precipitation, TCA/acetone precipitation, and phenol extraction combined with methanol/ammonium acetate precipitation, the latter showed to be the best method for black yeasts and meristematic fungi. Penicillium chrysogenum was used as reference strain.

Use of ribosomal introns as new markers of genetic diversity in Exophiala dermatitidis.

Exophiala dermatitidis is one of the prevalent black yeasts found as opportunistic pathogens or colonizers in humans. In the tropics its natural habitat is thought to be fruit surfaces and it is also found in the digestive system of fruit-eating animals. However, it has recently been abundantly isolated from human-made environments (steam baths, railway ties, dishwashers) in tropical and temperate climates. Two genotypes have been distinguished within this species: genotype A, mostly corresponding to strains isolated from patients, and genotype B, to strains isolated from the natural environment. In human-made environments, both genotypes A and B occur. A previous study suggested that one genotype had been selected for in the human host. In our study, the distribution of ribosomal insertions agrees with an ecological specialization of E. dermatitidis genotypes by showing a significantly higher frequency of ribosomal insertions in clinical strains in comparison to environmental ones. The characterization of these insertions shows that they correspond to introns of group IC or IE, the most frequent types within the fungal kingdom. These ribosomal group I introns could be used as new markers for populations of E. dermatitidis.

Cytotoxic metabolites from the soil-derived fungus Exophiala pisciphila.

A new polyketide compound 1 and a new naturally occurring chromone derivative 2, along with two known indole alkaloids 3-4 were characterized from the ethyl acetate extract of a soil-derived fungal strain, Exophiala pisciphila PHF-9. The structures of compounds 1-4 were established by detailed spectroscopic analysis and comparison with literature data. The absolute configuration of 1 was determined by a modified Mosher's method. Compound 1 exhibited moderate cytotoxicity against A-549, Hela, PANC-28 and BEL-7402 cell lines.

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.

Chlorohydroaspyrones A and B, antibacterial aspyrone derivatives from the marine-derived fungus Exophiala sp.

Chlorohydroaspyrones A (1) and B (2), antibacterial aspyrone derivatives, and the previously described aspyrone (3), asperlactone (4), and penicillic acid (5) have been isolated from the broth of a marine isolate of the fungus Exophiala. The structure and absolute stereochemistry of the compounds were determined on the basis of the physicochemical data analysis and chemical reactions. Compounds 1 and 2 exhibited a mild antibacterial activity against Staphylococcus aureus, methicillin-resistant S. aureus, and multidrug-resistant S. aureus. The MIC values of each strain are as follows: compound 1 showed 62.5 microg/mL for S. aureus and 125 microg/mL for methicillin-resistant S. aureus and multidrug-resistant S. aureus, and compound 2, 62.5 microg/mL for S. aureus and methicillin-resistant S. aureus and 125 microg/mL for multidrug-resistant S. aureus.

Circumdatin I, a new ultraviolet-A protecting benzodiazepine alkaloid from a marine isolate of the fungus Exophiala.

A new ultraviolet-A (UV-A) protecting benzodiazepine alkaloid, circumdatin I (1), along with the previously described circumdatin C (2) and circumdatin G (3), have been isolated from the mycelium of a marine-derived fungus of the genus Exophiala. The structures of the three circumdatins were assigned on the basis of physicochemical evidence. The absolute stereochemistry of 1 was determined by comparison of optical rotation and CD experiments with those of 2 and 3.

Isolation and characterization of a nitrile hydrolysing acidotolerant black yeast-Exophiala oligosperma R1.

Different nitriles were used as sole sources of nitrogen in a series of enrichments under acidic conditions to isolate acidotolerant nitriles hydrolysing microorganisms. From an enrichment in Na-citrate-phosphate buffer at pH 4 with glucose as carbon source and phenylacetonitrile as sole source of nitrogen, a black yeast (strain R1) was obtained which was identified by subsequent 18S rRNA gene sequencing as Exophiala oligosperma. The growth conditions of the organism were optimized for the production of cell material and the induction of the nitrile converting activity. Resting cell experiments demonstrated that phenylacetonitrile was converted via phenylacetic acid and 2-hydroxyphenylacetic acid. The organism could grow at pH 4 with phenylacetonitrile as sole source of carbon, nitrogen, and energy. The nitriles hydrolysing activity was also detected in cell-free extracts and indications for a nitrilase activity were found. The cell-free extracts converted, in addition to phenylacetonitrile, also different substituted phenylacetonitriles. Whole cells of E. oligosperma R1 converted phenylacetonitrile with almost the same reaction rates in the pH range from pH 1.5-pH 9.

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.