A Fasciclin Protein Is Essential for Laccase-Mediated Selective Phenol Coupling in Sporandol Biosynthesis.

The biaryl scaffold, often showing axial chirality, is a common feature of various fungal natural products. Their biosynthesis requires an oxidative phenol-coupling reaction usually catalyzed by laccases, cytochrome P450 enzymes, or peroxidases. The combination of a laccase and a fasciclin domain-containing (fas) protein is encoded in many biosynthetic gene clusters of biaryls from ascomycetes. However, such phenol-coupling systems including their regio- and stereoselectivity have not been characterized so far. Elucidating the biosynthesis of the antiparasitic binaphthalene sporandol from Chrysosporium merdarium, we demonstrate the combination of a laccase and a fas protein to be crucial for the dimerization reaction. Only the heterologous coproduction of the laccase and the fas protein led to a functional phenol-coupling system, whereas the laccase alone showed no coupling activity. Thus, the laccase/fas protein combination forms an independent group of phenol-coupling enzymes that determines the coupling activity and selectivity of the reaction concurrently and applies to the biosynthesis of many fungal natural products with a biaryl scaffold.

Molecular Diagnosis of Emmonsia-Like Fungi Occurring in Wild Animals.

Using specific primers based on the ribosomal operon, positive DNA amplification was obtained from lungs of 11/215 tested small burrowing animals, both terrestrial and aquatic, and including frozen (n = 4) and formalin-fixed paraffin-embedded (n = 7) samples. The main species detected in Europe in mice, otters and river rats was Emmonsia crescens. Two strains from otters and weasels were Blastomyces parvus. Two Australian wombats revealed the presence of a hitherto unknown species of the geophilic genus Emmonsiellopsis.

Disseminated Emergomycosis in a Person with HIV Infection, Uganda.

We describe emergomycosis in a patient in Uganda with HIV infection. We tested a formalin-fixed, paraffin-embedded skin biopsy to identify Emergomyces pasteurianus or a closely related pathogen by sequencing broad-range fungal PCR amplicons. Results suggest that emergomycosis is more widespread and genetically diverse than previously documented. PCR on tissue blocks may help clarify emergomycosis epidemiology.

Endangered Zoonotic Fungal Species from Chicken (Gallus gallus domestics).

Lophophyton gallinae (Microsporum gallinae) is a zoophilic fungus that causes ringworm in chickens and related species, and occasionally in humans. There are 45 human cases worldwide including a Japanese case from Okinawa in 2009. After the finding of the human L. gallinae case, 793 chickens in Japan, including 293 from the mainland and 500 from the Nansei Island areas, were investigated to determine the prevalence of dermatophytes and their related fungal species. The survey was carried out from December 2008 to March 2013. Various dermatophytes and related fungal species were isolated from the studied chickens, with a prevalence of 24.6%. In total, 224 dermatophytes and related species were isolated in the survey. The most commonly isolated species included, in descending order of frequency, Arthroderma multifidum, Aphanoascus terreus, and Chrysosporium spp. Ar. multifidum and Ap. terreus have no record of pathogenicity, and the present isolates of Chrysosporium spp. were not matched to pathogenic Chrysosporium spp. based on the ITS rDNA sequences. Interestingly, an L. gallinae isolate was detected in a male 10-month-old shamo (fighting cock) from the main island. Furthermore, one strain of Arthroderma simii was also isolated as the second record in Japan following that from an imported chimpanzee. Although L. gallinae and Ar. simii are likely to be endemic in our country, the transmission of dermatophytosis from chickens to humans is unlikely to occur because of the reduced chances for citizens to come in contact with chickens due to various factors.

Adiaspiromycosis in a wild European rabbit, and a review of the literature.

Adiaspiromycosis is a mycotic infection caused by thermally dimorphic fungi classified as Emmonsia parva and E. crescens (formerly Chrysosporium spp.) until recently, when new classifications were proposed. We document the pathologic findings in a severe case of adiaspiromycosis, with lymph node involvement, in a wild European rabbit ( Oryctolagus cuniculus). The rabbit exhibited granulomatous pneumonia with tracheobronchial lymph node enlargement. Histopathologically, the lung was expanded by myriad, densely cellular, heterophilic and granulomatous foci, surrounding bi- to trilaminar adiaspores. Adiaspore density was considered to be similar in all lung lobes. In the left caudal lung lobe, 80 adiaspores were counted in a 50-mm2 area using digital image analysis. The mean and median adiaspore diameters were 240 ± 52 µm and 255 µm, respectively. Tracheobronchial lymph nodes exhibited moderate numbers of similar adiaspores. PCR amplification of DNA extracted from microdissected adiaspores failed to identify Emmonsia spp.-specific DNA. These data suggest that adiaspiromycosis may result in severe granulomatous pneumonia in wild European rabbits. Although confirmation of the etiologic agent by PCR using DNA extracted from formalin-fixed tissue is not always successful, digital image analysis can be used to aid accurate assessment of adiaspore density and morphology.

Genomics and Comparative Genomic Analyses Provide Insight into the Taxonomy and Pathogenic Potential of Novel Emmonsia Pathogens.

Over the last 50 years, newly described species of Emmonsia-like fungi have been implicated globally as sources of systemic human mycosis (emmonsiosis). Their ability to convert into yeast-like cells capable of replication and extra-pulmonary dissemination during the course of infection differentiates them from classical Emmonsia species. Immunocompromised patients are at highest risk of emmonsiosis and exhibit high mortality rates. In order to investigate the molecular basis for pathogenicity of the newly described Emmonsia species, genomic sequencing and comparative genomic analyses of Emmonsia sp. 5z489, which was isolated from a non-deliberately immunosuppressed diabetic patient in China and represents a novel seventh isolate of Emmonsia-like fungi, was performed. The genome size of 5z489 was 35.5 Mbp in length, which is ~5 Mbp larger than other Emmonsia strains. Further, 9,188 protein genes were predicted in the 5z489 genome and 16% of the assembly was identified as repetitive elements, which is the largest abundance in Emmonsia species. Phylogenetic analyses based on whole genome data classified 5z489 and CAC-2015a, another novel isolate, as members of the genus Emmonsia. Our analyses showed that divergences among Emmonsia occurred much earlier than other genera within the family Ajellomycetaceae, suggesting relatively distant evolutionary relationships among the genus. Through comparisons of Emmonsia species, we discovered significant pathogenicity characteristics within the genus as well as putative virulence factors that may play a role in the infection and pathogenicity of the novel Emmonsia strains. Moreover, our analyses revealed a novel distribution mode of DNA methylation patterns across the genome of 5z489, with >50% of methylated bases located in intergenic regions. These methylation patterns differ considerably from other reported fungi, where most methylation occurs in repetitive loci. It is unclear if this difference is related to physiological adaptations of new Emmonsia, but this question warrants further investigation. Overall, our analyses provide a framework from which to further study the evolutionary dynamics of Emmonsia strains and identity the underlying molecular mechanisms that determine the infectious and pathogenic potency of these fungal pathogens, and also provide insight into potential targets for therapeutic intervention of emmonsiosis and further research.

In Vitro Antifungal Susceptibility of Yeast and Mold Phases of Isolates of Dimorphic Fungal Pathogen Emergomyces africanus (Formerly Emmonsia sp.) from HIV-Infected South African Patients.

Disseminated emmonsiosis is an important AIDS-related mycosis in South Africa that is caused by Emergomycesafricanus, a newly described and renamed dimorphic fungal pathogen. In vitro antifungal susceptibility data can guide management. Identification of invasive clinical isolates was confirmed phenotypically and by sequencing of the internal transcribed spacer region. Yeast and mold phase MICs of fluconazole, voriconazole, itraconazole, posaconazole, caspofungin, anidulafungin, micafungin, and flucytosine were determined with custom-made frozen broth microdilution (BMD) panels in accordance with Clinical and Laboratory Standards Institute recommendations. MICs of amphotericin B, itraconazole, posaconazole, and voriconazole were determined by Etest. Fifty unique E. africanus isolates were tested. The yeast and mold phase geometric mean (GM) BMD and Etest MICs of itraconazole were 0.01 mg/liter. The voriconazole and posaconazole GM BMD MICs were 0.01 mg/liter for both phases, while the GM Etest MICs were 0.001 and 0.002 mg/liter, respectively. The fluconazole GM BMD MICs were 0.18 mg/liter for both phases. The GM Etest MICs of amphotericin B, for the yeast and mold phases were 0.03 and 0.01 mg/liter. The echinocandins and flucytosine had very limited in vitro activity. Treatment and outcome data were available for 37 patients; in a multivariable model including MIC data, only isolation from blood (odds ratio [OR], 8.6; 95% confidence interval [CI], 1.3 to 54.4; P = 0.02) or bone marrow (OR, 12.1; 95% CI, 1.2 to 120.2; P = 0.03) (versus skin biopsy) was associated with death. In vitro susceptibility data support the management of disseminated emmonsiosis with amphotericin B, followed by itraconazole, voriconazole, or posaconazole. Fluconazole was a relatively less potent agent.

Novel taxa of thermally dimorphic systemic pathogens in the Ajellomycetaceae (Onygenales).

Recent discoveries of novel systemic fungal pathogens with thermally dimorphic yeast-like phases have challenged the current taxonomy of the Ajellomycetaceae, a family currently comprising the genera Blastomyces, Emmonsia, Emmonsiellopsis, Helicocarpus, Histoplasma, Lacazia and Paracoccidioides. Our morphological, phylogenetic and phylogenomic analyses demonstrated species relationships and their specific phenotypes, clarified generic boundaries and provided the first annotated genome assemblies to support the description of two new species. A new genus, Emergomyces, accommodates Emmonsia pasteuriana as type species, and the new species Emergomyces africanus, the aetiological agent of case series of disseminated infections in South Africa. Both species produce small yeast cells that bud at a narrow base at 37°C and lack adiaspores, classically associated with the genus Emmonsia. Another novel dimorphic pathogen, producing broad-based budding cells at 37°C and occurring outside North America, proved to belong to the genus Blastomyces, and is described as Blastomyces percursus.

Biodegradation of feather waste keratin by a keratinolytic soil fungus of the genus Chrysosporium and statistical optimization of feather mass loss.

This paper assesses the ability of strains of Aphanoascus fulvescens and Chrysosporium articulatum isolated from soil (phaesol) to degrade native feather keratin. Strains were identified based on phenotypic traits and nucleotide sequencing. Response Surface Methodology was used to optimize cultivation conditions exhibiting the highest keratinolytic activity. The experiments were based on Box-Behnken designs for the loss of substrate mass (chicken feathers). While substrate mass loss is an "economic coefficient" that reliably indicates feather keratin degradation, it has not been studied before. Stationary liquid cultures of five selected strains were conducted in laboratory conditions at 28 °C using poultry feathers (1 g) as the sole source of carbon, nitrogen and energy. Enzymatic activities, keratin mineralization products and substrate mass loss were determined periodically. The mineralization of keratin proteins by strains yielded a high number of ammonium ions alkalinizing the medium. Increased ammonium ions inhibited the activity of caseinian protease and keratinase. A decrease in the concentration of these ions induced proteolytic enzymes, chiefly the activity of keratinase, at the end of fungal cultivation. Keratinase activity was related to protein- and peptide release and that of caseinian protease to sulfate ions. The highest loss of substrate mass in comparison to the reference strain CBS104.62 (35.4%) was recorded for Aphanoascus fulvescens B21/4-5 (65.9%). Based on a Box-Behnken design, the maximum loss of substrate mass for the Aphanoascus fulvescens strain (71.08%) can be achieved at pH 7.58 and temperature 28.7 °C.

Snake fungal disease caused by Ophidiomyces ophiodiicola in a free-ranging mud snake (Farancia abacura).

Snake fungal disease is an emerging infectious disease caused by the fungus Ophidiomyces ophiodiicola leading to severe dermatitis and facial disfiguration in numerous free-ranging and captive snakes. A free-ranging mud snake (Farancia abacura) from Bulloch County, Georgia, was presented for autopsy because of facial swelling and emaciation. Extensive ulceration of the skin, which was especially severe on the head, and retained shed were noted on external examination. Microscopic examination revealed severe heterophilic dermatitis with intralesional fungal hyphae and arthroconidia consistent with O. ophiodiicola A skin sample incubated on Sabouraud dextrose agar yielded a white-to-tan powdery fungal culture that was confirmed to be O. ophiodiicola by polymerase chain reaction and sequence analysis. Heavy infestation with adult tapeworms (Ophiotaenia faranciae) was present within the intestine. Various bacterial and fungal species, interpreted to either be secondary invaders or postmortem contaminants, were associated with oral lesions. Although the role of these other organisms in the overall health of this individual is not known, factors such as concurrent infections or immunosuppression should be considered in order to better understand the overall manifestation of snake fungal disease, which remains poorly characterized in its host range and geographic distribution.

Isolation and Characterization of an Unknown Chrysosporium sp. Producing Subcutaneous Mycosis in an Immunocompromised Patient.

We report a case of subcutaneous fungal abscess over the great toe caused by a keratinophilic fungus, an unknown Chrysosporium sp., in a 60-year-old diabetic female who was treated successfully with oral fluconazole. The fungus was isolated from aspirated pus, and septate hyphae were seen in fine needle aspiration cytology. Ovoid- to club-shaped hyaline one-celled conidia (aleuriconidia) with broad truncated bases were seen, and sequencing of ITS1-5.8S-ITS2 rDNA revealed belonging to the order Onygenales and most closely related to Chrysosporium spp. isolated from a fowl. Of the 65 species within the genus Chrysosporium, very few have been reported as pathogenic.

Disseminated Emmonsia pasteuriana infection in India: a case report and a review.

We report here the first case of disseminated Emmonsia pasteuriana infection in a patient with AIDS in India. The patient presented with weight loss, dyspnoea, left-sided chest pain and multiple non-tender skin lesions over face and body for 3 months. Disseminated emmonsiosis was diagnosed on microscopic examination and fungal culture of skin biopsy and needle aspirate of lung consolidation. It was confirmed by sequencing internal transcribed spacer region of rDNA, beta tubulin, actin, and intein PRP8. The patient responded to amphotericin B and itraconazole therapy.

The Dynamic Genome and Transcriptome of the Human Fungal Pathogen Blastomyces and Close Relative Emmonsia.

Three closely related thermally dimorphic pathogens are causal agents of major fungal diseases affecting humans in the Americas: blastomycosis, histoplasmosis and paracoccidioidomycosis. Here we report the genome sequence and analysis of four strains of the etiological agent of blastomycosis, Blastomyces, and two species of the related genus Emmonsia, typically pathogens of small mammals. Compared to related species, Blastomyces genomes are highly expanded, with long, often sharply demarcated tracts of low GC-content sequence. These GC-poor isochore-like regions are enriched for gypsy elements, are variable in total size between isolates, and are least expanded in the avirulent B. dermatitidis strain ER-3 as compared with the virulent B. gilchristii strain SLH14081. The lack of similar regions in related species suggests these isochore-like regions originated recently in the ancestor of the Blastomyces lineage. While gene content is highly conserved between Blastomyces and related fungi, we identified changes in copy number of genes potentially involved in host interaction, including proteases and characterized antigens. In addition, we studied gene expression changes of B. dermatitidis during the interaction of the infectious yeast form with macrophages and in a mouse model. Both experiments highlight a strong antioxidant defense response in Blastomyces, and upregulation of dioxygenases in vivo suggests that dioxide produced by antioxidants may be further utilized for amino acid metabolism. We identify a number of functional categories upregulated exclusively in vivo, such as secreted proteins, zinc acquisition proteins, and cysteine and tryptophan metabolism, which may include critical virulence factors missed before in in vitro studies. Across the dimorphic fungi, loss of certain zinc acquisition genes and differences in amino acid metabolism suggest unique adaptations of Blastomyces to its host environment. These results reveal the dynamics of genome evolution and of factors contributing to virulence in Blastomyces.

Disseminated infection caused by Emmonsia pasteuriana in a renal transplant recipient.

Emmonsia pasteuriana is a thermally dimorphic fungus identified in very few human cases. Here, we report a case of a 43-year-old male renal transplant patient from China presenting with multiple painful skin eruptions on his head, nose and left thigh, later accompanied by respiratory failure. Histopathology of the biopsy collected from the left thigh upper ulcer and occipital nodule both demonstrated chronic inflammation with granuloma formation and yeast-like elements. Emmonsia pasteuriana was cultured from two biopsy specimens and their identity was confirmed by sequencing of the rDNA internal transcribed spacer. The patient in intensive care showed marked clinical improvement with antifungal treatment.

TaqMan real-time polymerase chain reaction for detection of Ophidiomyces ophiodiicola, the fungus associated with snake fungal disease.

BACKGROUND: Fungal skin infections associated with Ophidiomyces ophiodiicola, a member of the Chrysosporium anamorph of Nannizziopsis vriesii (CANV) complex, have been linked to an increasing number of cases of snake fungal disease (SFD) in captive snakes around the world and in wild snake populations in eastern North America. The emergence of SFD in both captive and wild situations has led to an increased need for tools to better diagnose and study the disease. RESULTS: We developed two TaqMan real-time polymerase chain reaction (PCR) assays to rapidly detect O. ophiodiicola in clinical samples. One assay targets the internal transcribed spacer region (ITS) of the fungal genome while the other targets the more variable intergenic spacer region (IGS). The PCR assays were qualified using skin samples collected from 50 snakes for which O. ophiodiicola had been previously detected by culture, 20 snakes with gross skin lesions suggestive of SFD but which were culture-negative for O. ophiodiicola, and 16 snakes with no clinical signs of infection. Both assays performed equivalently and proved to be more sensitive than traditional culture methods, detecting O. ophiodiicola in 98% of the culture-positive samples and in 40% of the culture-negative snakes that had clinical signs of SFD. In addition, the assays did not cross-react with a panel of 28 fungal species that are closely related to O. ophiodiicola or that commonly occur on the skin of snakes. The assays did, however, indicate that some asymptomatic snakes (~6%) may harbor low levels of the fungus, and that PCR should be paired with histology when a definitive diagnosis is required. CONCLUSIONS: These assays represent the first published methods to detect O. ophiodiicola by real-time PCR. The ITS assay has great utility for assisting with SFD diagnoses whereas the IGS assay offers a valuable tool for research-based applications.

Invasive pulmonary infection caused by Chrysosporium articulatum: the first case report.

Chrysosporium species, saprobic soil fungi, comprise more than 60 species. There is some confusion regarding the taxonomy and nomenclature between Chrysosporium and Emmonsia since the causative agents of adiaspiromycosis, the development of big thick-walled spores (adiaspores) in humans or animals, were previously thought to be Chrysosporium. Chrysosporium articulatum has never been reported to cause invasive infection in humans. We report herein the first case of invasive pulmonary infection caused by Chrysosporium articulatum in a 16-year-old man with acute T-cell lymphoblastic leukaemia. He was successfully treated with voriconazole.

A dimorphic fungus causing disseminated infection in South Africa.

BACKGROUND: The genus emmonsia contains three species that are associated with human disease. Emmonsia crescens and Emmonsia parva are the agents that cause adiaspiromycosis, and one human case of Emmonsia pasteuriana infection has been described. We report a fungal pathogen within the genus emmonsia that is most closely related to E. pasteuriana in human immunodeficiency virus (HIV)-infected adults in South Africa. METHODS: Between July 2008 and July 2011, we conducted enhanced surveillance to identify the cause of systemic, dimorphic fungal infections in patients presenting to Groote Schuur Hospital and other hospitals affiliated with the University of Cape Town, Cape Town, South Africa. DNA sequencing was used to identify pathogenic fungi. RESULTS: A total of 24 cases of dimorphic fungal infection were diagnosed, 13 of which were caused by an emmonsia species. All 13 patients were HIV-infected, with a median CD4+ T-cell count of 16 cells per cubic millimeter (interquartile range, 10 to 44), and all had evidence of disseminated fungal disease. Three patients died soon after presentation, but the others had a good response to a variety of antifungal agents and antiretroviral therapy. Phylogenetic analysis of five genes (LSU, ITS1-2, and the genes encoding actin, ß-tubulin, and intein PRP8) revealed that this fungus belongs in the genus emmonsia and is most closely related to E. pasteuriana. CONCLUSIONS: The findings suggest that these isolates of an emmonsia species represent a new species of dimorphic fungus that is pathogenic to humans. The species appears to be an important cause of infections in Cape Town.

Molecular characterization of reptile pathogens currently known as members of the chrysosporium anamorph of Nannizziopsis vriesii complex and relationship with some human-associated isolates.

In recent years, the Chrysosporium anamorph of Nannizziopsis vriesii (CANV), Chrysosporium guarroi, Chrysosporium ophiodiicola, and Chrysosporium species have been reported as the causes of dermal or deep lesions in reptiles. These infections are contagious and often fatal and affect both captive and wild animals. Forty-nine CANV isolates from reptiles and six isolates from human sources were compared with N. vriesii based on their cultural characteristics and DNA sequence data. Analyses of the sequences of the internal transcribed spacer and small subunit of the nuclear ribosomal gene revealed that the reptile pathogens and human isolates belong in well-supported clades corresponding to three lineages that are distinct from all other taxa within the family Onygenaceae of the order Onygenales. One lineage represents the genus Nannizziopsis and comprises N. vriesii, N. guarroi, and six additional species encompassing isolates from chameleons and geckos, crocodiles, agamid and iguanid lizards, and humans. Two other lineages comprise the genus Ophidiomyces, with the species Ophidiomyces ophiodiicola occurring only in snakes, and Paranannizziopsis gen. nov., with three new species infecting squamates and tuataras. The newly described species are Nannizziopsis dermatitidis, Nannizziopsis crocodili, Nannizziopsis barbata, Nannizziopsis infrequens, Nannizziopsis hominis, Nannizziopsis obscura, Paranannizziopsis australasiensis, Paranannizziopsis californiensis, and Paranannizziopsis crustacea. Chrysosporium longisporum has been reclassified as Paranannizziopsis longispora. N. guarroi causes yellow fungus disease, a common infection in bearded dragons and green iguanas, and O. ophiodiicola is an emerging pathogen of captive and wild snakes. Human-associated species were not recovered from reptiles, and reptile-associated species were recovered only from reptiles, thereby mitigating concerns related to zoonosis.