EpABC Genes in the Adaptive Responses of Exophiala pisciphila to Metal Stress: Functional Importance and Relation to Metal Tolerance.

Exophiala pisciphila is one of the dominant dark septate endophytes (DSEs) colonizing metal-polluted slag heaps in southwest China. It shows numerous super-metal-tolerant characteristics, but the molecular mechanisms involved remain largely unknown. In the present study, the functional roles of a specific set of ATP-binding cassette (ABC) transporters in E. pisciphila were characterized. In total, 26 EpABC genes belonging to 6 subfamilies (ABCA to ABCG) were annotated in previous transcriptome sequencing libraries, and all were regulated by metal ions (Pb, Zn, and Cd), which was dependent on the metal species and/or concentrations tested. The results from the heterologous expression of 3 representative EpABC genes confirmed that the expression of EpABC2.1, EpABC3.1, or EpABC4.1 restored the growth of metal-sensitive mutant Saccharomyces cerevisiae strains and significantly improved the tolerance of Arabidopsis thaliana to Pb, Zn, and Cd. Interestingly, the expression of the 3 EpABC genes further altered metal (Pb, Zn, and Cd) uptake and accumulation and promoted growth by alleviating the inhibitory activity in yeast and thale cress caused by toxic ions. These functions along with their vacuolar location suggest that the 3 EpABC transporters may enhance the detoxification of vacuolar compartmentation via transport activities across their membranes. In conclusion, the 26 annotated EpABC transporters may play a major role in maintaining the homeostasis of various metal ions in different cellular compartments, conferring an extreme adaptative advantage to E. pisciphila in metal-polluted slag heaps.IMPORTANCE Many ABC transporters and their functions have been identified in animals and plants. However, little is known about ABC genes in filamentous fungi, especially DSEs, which tend to dominantly colonize the roots of plants growing in stressed environments. Our results deepen the understanding of the function of the ABC genes of a super-metal-tolerant DSE (E. pisciphila) in enhancing its heavy metal resistance and detoxification. Furthermore, the genetic resources of DSEs, e.g., numerous EpABC genes, especially from super-metal-tolerant strains in heavy metal-polluted environments, can be directly used for transgenic applications to improve tolerance and phytoextraction potential.

Ecology of the Human Opportunistic Black Yeast Exophiala dermatitidis Indicates Preference for Human-Made Habitats.

Exophiala dermatitidis is an ascomycetous black yeast from the order Chaetothyriales. Its growth characteristics include the polymorphic life cycle, ability to grow at high and low temperatures, at a wide pH range, survival at high concentrations of NaCl, and survival at high UV and radioactive radiation. Exophiala dermatitidis causes deep or localized phaeohyphomycosis in immuno-compromised people worldwide and is regularly encountered in the lungs of cystic fibrosis patients. Regardless of numerous ecological studies worldwide, little is known about its natural habitat or the possible infection routes. The present review summarizes the published data on its frequency of occurrence in nature and in man-made habitats. We additionally confirmed its presence with culture-depending methods from a variety of habitats, such as glacial meltwater, mineral water, mineral-rich salt-pan mud, dishwashers, kitchens and different environments polluted with aromatic hydrocarbons. In conclusion, the frequency of its recovery was the highest in man-made indoor habitats, connected to water sources, and exposed to occasional high temperatures and oxidative stress.

Is Marine Dispersion of the Lethargic Crab Disease Possible? Assessing the Tolerance of Exophiala cancerae to a Broad Combination of Salinities, Temperatures, and Exposure Times.

Since 1997, an emergent fungal disease named lethargic crab disease (LCD) has decimated stocks of the edible mangrove land crab Ucides cordatus (Linnaeus, 1763) (Brachyura: Ocypodidae) along the Brazilian coast, threatening the mangrove ecosystem and causing socioeconomic impacts. Evidence from a variety of sources suggests that the black yeast Exophiala cancerae (Herpotrichiellaceae, Chaetothyriales) has been responsible for such epizootic events. Based on the spatiotemporal patterns of the LCD outbreaks, the well-established surface ocean currents, and the range of ecological traits of Exophiala spp., a marine dispersal hypothesis may be proposed. Using in vitro experiments, we tested the survival and growth of E. cancerae CBS 120420 in a broad combination of salinities, temperatures, and exposure times. While variation in salinity did not significantly affect the growth of colony-forming units (CFUs) (P > 0.05), long exposure times visibly influenced an increase in CFUs growth (P < 0.05). However, higher temperature (30 °C) caused a reduction of about 1.2-fold in CFUs growth (P < 0.05). This result suggests that sea surface temperatures either above or below the optimum growth range of E. cancerae could play a key role in the apparent north-south limits in the geographical distribution of LCD outbreaks. In light of our results, we conclude that a fundamental step toward the understanding of LCD epidemiological dynamics should comprise a systematic screening of E. cancerae in estuarine and coastal waters.

Combination of Amphotericin B and Flucytosine against Neurotropic Species of Melanized Fungi Causing Primary Cerebral Phaeohyphomycosis.

Primary central nervous system phaeohyphomycosis is a fatal fungal infection due mainly to the neurotropic melanized fungiCladophialophora bantiana,Rhinocladiella mackenziei, andExophiala dermatitidis.Despite the combination of surgery with antifungal treatment, the prognosis continues to be poor, with mortality rates ranging from 50 to 70%. Therefore, a search for a more-appropriate therapeutic approach is urgently needed. Ourin vitrostudies showed that with the combination of amphotericin B and flucytosine against these species, the median fractional inhibitory concentration (FIC) indices for strains ranged from 0.25 to 0.38, indicating synergy. By use of Bliss independence analysis, a significant degree of synergy was confirmed for all strains, with the sum ΔE ranging from 90.2 to 698.61%. No antagonism was observed. These results indicate that amphotericin B, in combination with flucytosine, may have a role in the treatment of primary cerebral infections caused by melanized fungi belonging to the orderChaetothyriales Furtherin vivostudies and clinical investigations to elucidate and confirm these observations are warranted.

Heterotrophic Bioleaching of Sulfur, Iron, and Silicon Impurities from Coal by Fusarium oxysporum FE and Exophiala spinifera FM with Growing and Resting Cells.

Coal is the most abundant fossil fuel containing sulfur and other elements which promote environmental pollution after burning. Also the silicon impurities make the transportation of coal expensive. In this research, two isolated fungi from oil contaminated soil with accessory number KF554100 (Fusarium oxysporum FE) and KC925672 (Exophiala spinifera FM) were used for heterotrophic biological leaching of coal. The leaching were detected by FTIR, CHNS, XRF analyzer and compared with iron and sulfate released in the supernatant. The results showed that E. spinifera FM produced more acidic metabolites in growing cells, promoting the iron and sulfate ions removal while resting cells of F. oxysporum FE enhanced the removal of aromatic sulfur. XRF analysis showed that the resting cells of E. spinifera FM proceeded maximum leaching for iron and silicon (48.8, 43.2 %, respectively). CHNS analysis demonstrated that 34.21 % of sulfur leaching was due to the activities of resting cells of F. oxysporum FE. Also F. oxysporum FE removed organic sulfur more than E. spinifera FM in both growing and resting cells. FTIR data showed that both fungi had the ability to remove pyrite and quartz from coal. These data indicated that inoculations of these fungi to the coal are cheap and impurity removals were faster than autotrophic bacteria. Also due to the removal of dibenzothiophene, pyrite, and quartz, we speculated that they are excellent candidates for bioleaching of coal, oil, and gas.

Shared Physiological Traits of Exophiala Species in Cold-Blooded Vertebrates, as Opportunistic Black Yeasts.

Several species of the genus Exophiala are found as opportunistic pathogens on humans, while others cause infections in cold-blooded waterborne vertebrates. Opportunism of these fungi thus is likely to be multifactorial. Ecological traits [thermotolerance and pH tolerance, laccase activity, assimilation of mineral oil, and decolorization of Remazol Brilliant Blue R (RBBR)] were studied in a set of 40 strains of mesophilic Exophiala species focused on the salmonis-clade mainly containing waterborne species. Thermophilic species and waterborne species outside the salmonis-clade were included for comparison. Strains were able to tolerate a wide range of pHs, although optimal growth was observed between pH 4.0 and 5.5. All strains tested were laccase positive. Strains were able to grow in the presence of the compounds (mineral oil and RBBR) with some differences in assimilation patterns between strains tested and also were capable of degrading the main chromophore of RBBR. The study revealed that distantly related mesophilic species behave similarly, and no particular trend in evolutionary adaptation was observed.

Effects of Tricyclazole on Cadmium Tolerance and Accumulation Characteristics of a Dark Septate Endophyte (DSE), Exophiala pisciphila.

Exophiala pisciphila is a cadmium-tolerant fungus, and produces 1,8-dihydroxynaphthalene melanin which can be inhibited by tricyclazole. Tricyclazole at higher levels (20 and 40 µg mL−1) reduced the growth and sporulation of E. pisciphila, but toxicity was not observed at a low concentration (2.5 µg mL−1). Under cadmium (Cd) stress (50, 100 and 200 mg L−1), 2.5 µg mL−1 of tricyclazole reduced fungal growth and sporulation. These reduces indicated a decrease on Cd tolerance of E. pisciphila. For both the 0 and 2.5 µg mL−1 tricyclazole treatments, Cd was associated mostly with cell walls and was extracted by 2 % acetic acid and 1 M NaCl. The FTIR spectra of the E. pisciphila mycelia were similar for both 0 and 2.5 µg mL−1 tricyclazole treatments, which showed hydroxyl, amine, carboxyl and phosphate groups. Thus inhibition of melanin synthesis by tricyclazole did not change Cd accumulation characteristics in E. pisciphila. Results suggested that melanin played a protective role for E. pisciphila against Cd stress, but inhibition of melanin synthesis did not have a remarkable impact on Cd accumulation in E. pisciphila.

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.

Massive contamination of Exophiala dermatitidis and E. phaeomuriformis in railway stations in subtropical Turkey.

In order to reveal the source of contamination of opportunistic fungi, their natural habitat has to be understood. Black yeast-like fungi are abundant in man-made environments, particularly in those that are rich in toxic hydrocarbons such as railway ties. In this study, we investigated the presence of black fungi on creosote-treated oak railway ties and concrete sleepers stained with petroleum oil. Samples were collected at two central stations in Turkish cities, Mersin and Adana, and from Tarsus town station located between these two. The sample locations had subtropical climates. A total of 570 railway samples, including 320 from oak and 250 from concrete, were collected. Cotton swabs moistened with sterile physiological saline were applied to the ties and inoculated onto malt extract agar followed by incubation at 37 °C. Overall, we recovered 97 black yeast-like fungi (17.0 % positive). Sixty-three fungi (19.7 %) were collected from creosote-treated oak, whereas 34 isolates (13.6 %) were derived from concrete; the difference was significant (P = 0.05). Identification using rDNA internal transcribed spacer revealed Exophiala dermatitidis (57.7 %) and Exophiala phaeomuriformis (42.3 %). This study suggested that hydrocarbons enrich these opportunistic black yeasts. An eventual health risk is discussed.

Identification of Hortaea werneckii Isolated from mangrove plant Aegiceras comiculatum based on morphology and rDNA sequences.

Hortaea werneckii is a black yeast-like ascomycetous fungi associated with the human superficial infection tinea nigra, which commonly occurs in tropical and subtropical countries. Now, this fungus has been found in the halophilic environment all over the world and recognized as a new model organism in exploring the mechanisms of salt tolerance in eukaryotes. During a survey of endophytic fungi of mangrove forest at South China Sea, two isolates of H. werneckii were recovered from medicinal plant of Aegiceras comiculatum. The isolates were identified by morphological characters and phylogenetic analyses (e.g., ITS rDNA, LSU rDNA and translation elongation factor EF1α). Some physiological tests such as thermotolerance, acid tolerance (pH) and NaCl tolerance as well as pathogenicity test in vitro for the strains of Hortaea were performed. It is the first report that H. werneckii was isolated from medicinal plant of A. comiculatum in south sea of China as the endophytic fungi.

[Synthesis of melanin pigments by Antarctic black yeast].

Five strains of the black yeast similar to Exophiala nigra (Nadsoniela nigra), which we have isolated from the Antarctic biotopes, are studied. At cultivation in a periodic operation the maximum level of absolutely dry biomass in five tested strains constituted 3.2-7.8 g/l of medium, melanin pigment yield being 6-9% of absolutely dry mass of cells. Two highly productive strains have been selected. Pigments of the studied black yeast are water-insoluble, however dissolve in alkali and concentrated acids. The maximum absorption of the yeast pigments was in the range of 220 nm. The above-stated properties of pigments of the investigated yeast correspond to the description of melanin fractions of Nadsoniela nigra and some microscopic mushrooms. The water-soluble melanin-pigments have been obtained after the dialysis of alkaline solution of the pigment. UV-spectra and visible absorption spectra of water solution of melanin-pigments are almost identical to those of initial alkaline solutions. It is shown that the studied yeast are resistant to high concentrations of toxic metals (Hg2+, Cu2+, Co2+, Cr(VI) and Ni2+), and introduction of Co2+ into the cultivation medium leads to the increase of pigments synthesis.

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.

Phaeohyphomycosis of the face caused by Exophiala oligosperma.

A 57-year-old previously healthy woman who works in the fish-processing industry presented with a 1-year history of a slightly pruritic, hyperkeratotic, brownish, erythematous lesion of the left cheek measuring 5 × 5 mm in diameter. Histopathology revealed granuloma formation in the superficial dermal layer by multinucleated giant cells that contained pale-brown septate hyphae. Periodic acid-Schiff stain showed many hyphae and catenate spores within the multinucleated giant cells. Tissue specimens and skin scrapings were obtained and incubated on mycosel agar, yielding black, velvety colonies that were morphologically identified as belonging to Exophiala species. Sequence analysis of the internal transcribed spacer region of the ribosomal RNA gene showed 99-100% homology to Exophiala oligosperma sequences. This report describes a rare case of phaeohyphomycosis of the face caused by E. oligosperma.

Bioconversion of 2,6-dimethylpyridine to 6-methylpicolinic acid by Exophiala dermatitidis (Kano) de Hoog DA5501 cells grown on n-dodecane.

Alkane-assimilating microorganisms were isolated from enrichment cultures using n-octane, n-dodecane, n-hexadecane, or pristane (2,6,10,14-tetramethylpentadecane) as a sole carbon source to find microbial catalysts oxidizing methyl groups of 2,6-dimethylpyridine. The cells of Exophiala dermatitidis (Kano) de Hoog DA5501, an n-dodecane-assimilating fungus, oxidized a single methyl group of 2,6-dimethylpyridine to produce 6-methylpicolinic acid (6-methylpyridine-2-carboxylic acid) without the formation of dipicolinic acid (pyridine-2,6-dicarboxylic acid); 67 mM 6-methylpicolinic acid (9.2 g/l) accumulated with a molar conversion yield of 89% by 54-h incubation. The fungus cells also oxidized the methyl group of 2,6-dimethylpyrazine and 2,4,6-trimethylpyridine regioselectively.

[Resistance to UV radiation of microorganisms isolated from the rock biotopes of the Antarctic region].

Microbiological analysis of terrestrial biotopes of the Antarctic Region has shown, that vertical rocks of the Antarctic islands open for the Sun were characterized by special microcenoses. The wide distribution of pigmented microorganisms in the rock Antarctic samples, a higher frequency of their occurrence, the total number and biologic diversity, than in other Antarctic biotopes, has been demonstrated. For the first time the presence of bacteria and yeast, resistant to high doses of UV radiation on the vertical rocks in the Antarctic Region was shown. The lethal doze of UV radiation for the Antarctic pink pigmented Methylobacterium strains exceeded 200-300 J/m2, for coal-black yeast--500-800 J/m2, for red yeast--1200-1500 J/m2. The distinctions in lethal UV effect against strains of Methylobacterium isolated from the regions with different climate have not been found. Probably, adaptation of the rock microcenosis to extreme factors of the environment proceeds by natural selection of microorganisms, which resistance to this factor is genetically determined.

[Screening of yeast-producers of melanin in the Antarctic terrestrial biotopes].

Now it is considered, that melanin is promising for application in medicine and pharmacology. Since black yeast were found in polar regions before, we have carried out screening of yeast-producers of melanin in the Antarctic biotopes. Dark pigmented microorganisms are revealed in 30% of samples from terrestrial biotopes at the western coast of the Antarctic peninsula, on islands of the Argentina archipelago (Galindez, Skua, Corner, Barchans, Irizar, Uruguay, Cruls, Three little pigs, King-George), and also on the neighbouring islands Petermann, Jalour, Berthelot, Darboux and Lippmann. In the Antarctic lichens the occurrence frequency of dark pigmented microorganisms, their total number and biodiversity are significantly higher, than in other Antarctic biotopes. Coal-black yeast are found on the crustose and bushy lichens on vertical rocks, they occur less often in soil, their quantity made 1 x 10(2) - 6 x 10(3)/g of a sample. Coal-black pigments were isolated from two Antarctic strains of yeast. These pigments are identical to melanin according to a complex of specific chemical tests, that is also confirmed by the character of UV-spectra (220-230 nm) and absorption spectra in the visible area (400-800 nm). The output of synthesized pigment/g in strain 36 made more than 10% of biomass amount. So, the yeast synthesizing intensively melanin are revealed in the Antarctic terrestrial biotopes.

Radioadapted Wangiella dermatitidis senses radiation in its environment in a melanin-dependent fashion.

Black fungi withstand extreme stresses partly due to the presence of melanin. Melanin is associated with structural integrity and resistance to chemical and radiation stress. This results in improved health and fitness, specifically in extreme conditions. Our goal was to exploit the radiation sensing nature of melanized fungus in order to develop a radioadapted strain capable of responding to radiation in the environment. The protracted exposure of a melanized fungus, Wangiella dermatitidis, to a mixed source of radiation altered the electron transport properties. There was no effect in an albino mutant wdpsk1. We then tested the growth response to radiation in the environment, with shielding from direct exposure to the radiation. Gamma radiation caused increased colony growth irrespective of exposure history in melanized fungus. Beta particles produced growth inhibition. The previously exposed melanized strain demonstrated colony growth in response to alpha particles in the environment. Alpha particles have a higher linear energy transfer, which produces more reactive oxygen species. Our previously exposed melanized strain was resistant to the toxic effects of H2O2, while the naïve and non-melanized strains were sensitive. We propose that previous radiation exposure introduces adaptations that equip melanized fungi to tolerate, sense, and respond to radiation byproducts.

Proteomics Reveals Distinct Changes Associated with Increased Gamma Radiation Resistance in the Black Yeast Exophiala dermatitidis.

The yeast Exophiala dermatitidis exhibits high resistance to γ-radiation in comparison to many other fungi. Several aspects of this phenotype have been characterized, including its dependence on homologous recombination for the repair of radiation-induced DNA damage, and the transcriptomic response invoked by acute γ-radiation exposure in this organism. However, these findings have yet to identify unique γ-radiation exposure survival strategies-many genes that are induced by γ-radiation exposure do not appear to be important for recovery, and the homologous recombination machinery of this organism is not unique compared to more sensitive species. To identify features associated with γ-radiation resistance, here we characterized the proteomes of two E. dermatitidis strains-the wild type and a hyper-resistant strain developed through adaptive laboratory evolution-before and after γ-radiation exposure. The results demonstrate that protein intensities do not change substantially in response to this stress. Rather, the increased resistance exhibited by the evolved strain may be due in part to increased basal levels of single-stranded binding proteins and a large increase in ribosomal content, possibly allowing for a more robust, induced response during recovery. This experiment provides evidence enabling us to focus on DNA replication, protein production, and ribosome levels for further studies into the mechanism of γ-radiation resistance in E. dermatitidis and other fungi.

Liver cirrhosis caused by Exophiala dermatitidis.

We report a case of liver cirrhosis caused by Exophiala dermatitidis in a previously healthy child. The infecting organism was initially mistaken as capsule-deficient Cryptococcus neoformans.

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.