Ajellomyces Dermatitidis Research Articles

Structure of the imine reductase from Ajellomyces dermatitidis in three crystal forms.

The NADPH-dependent imine reductase from Ajellomyces dermatitidis (AdRedAm) catalyzes the reductive amination of certain ketones with amine donors supplied in an equimolar ratio. The structure of AdRedAm has been determined in three forms. The first form, which belongs to space group P3121 and was refined to 2.01 Å resolution, features two molecules (one dimer) in the asymmetric unit in complex with the redox-inactive cofactor NADPH4. The second form, which belongs to space group C21 and was refined to 1.73 Å resolution, has nine molecules (four and a half dimers) in the asymmetric unit, each complexed with NADP+. The third form, which belongs to space group P3121 and was refined to 1.52 Å resolution, has one molecule (one half-dimer) in the asymmetric unit. This structure was again complexed with NADP+ and also with the substrate 2,2-difluoroacetophenone. The different data sets permit the analysis of AdRedAm in different conformational states and also reveal the molecular basis of stereoselectivity in the transformation of fluorinated acetophenone substrates by the enzyme.

Synthesis of 2,5‐Disubstituted Pyrrolidine Alkaloids via A One‐Pot Cascade Using Transaminase and Reductive Aminase Biocatalysts

AbstractA multi‐enzymatic cascade process involving transaminases (TAs) and reductive aminases (RedAms) to produce enantiomerically pure 2,5‐disubstituted pyrrolidine alkaloids from their respective 1,4‐diketones is reported. Several TAs were screened and the best results for diketone monoamination were obtained with an R‐selective TA from Mycobacterium chlorophenicum and with an S‐selective TA from Bacillus megaterium. Pyrroline reduction was best performed by a reductive aminase from Ajellomyces dermatitidis (AdRedAm). Finally, a biocatalytic one‐pot cascade was implemented using the aforementioned enzymes and a variety of 2‐methyl‐5‐alkylpyrrolidines were produced with high (>99 %) conversion, diastereomeric and enantiomeric excess values.

Fungal dimorphism: the switch from hyphae to yeast is a specialized morphogenetic adaptation allowing colonization of a host.

The ability of pathogenic fungi to switch between a multicellular hyphal and unicellular yeast growth form is a tightly regulated process known as dimorphic switching. Dimorphic switching requires the fungus to sense and respond to the host environment and is essential for pathogenicity. This review will focus on the role of dimorphism in fungi commonly called thermally dimorphic fungi, which switch to a yeast growth form during infection. This group of phylogenetically diverse ascomycetes includes Talaromyces marneffei (recently renamed from Penicillium marneffei), Blastomyces dermatitidis (teleomorph Ajellomyces dermatitidis), Coccidioides species (C. immitis and C. posadasii), Histoplasma capsulatum (teleomorph Ajellomyces capsulatum), Paracoccidioides species (P. brasiliensis and P. lutzii) and Sporothrix schenckii (teleomorph Ophiostoma schenckii). This review will explore both the signalling pathways regulating the morphological transition and the transcriptional responses necessary for intracellular growth. The physiological requirements of yeast cells during infection will also be discussed, highlighting recent advances in the understanding of the role of iron and calcium acquisition during infection.

Granulomatous rhinitis due to Candida parapsilosis in a cat

A 9-year-old female spayed Domestic Medium Hair cat presented to the referring veterinarian with a 2-week history of sneezing, which progressed to swelling over the nasal planum. The cat had been under veterinary care for inflammatory bowel disease and had been treated with 1.25 mg/kg prednisolone once a day for approximately 1 year. On physical examination, an approximately 2-3 mm diameter, round polypoid pink soft-tissue mass was protruding slightly from the right nostril. Through histologic examination of representative sections from the mass, there was a severe diffuse infiltrate of epithelioid macrophages and neutrophils that surrounded frequent 15-20 µm yeast organisms. A Grocott methenamine silver stain revealed the presence of pseudohyphae in addition to the previously noted yeast forms. Real-time polymerase chain reaction (PCR) for Cryptococcus neoformans, Ajellomyces dermatitidis (syn. Blastomyces dermatitidis), Coccidioides immitis, Ajellomyces capsulatus (syn. Histoplasma capsulatum), Malassezia spp., and Candida spp. was performed on the paraffin-embedded sample. The PCR for Candida spp. was positive; the product was then sequenced and was determined to be consistent with Candida parapsilosis. Following the PCR diagnosis and prior to treatment of the infection, C. parapsilosis was cultured from a nasal swab. The infection in the cat in the current report was considered opportunistic and secondary to immunosuppression, following treatment for the inflammatory bowel disease.

Identification of the Mating-Type (MAT) Locus That Controls Sexual Reproduction of Blastomyces dermatitidis

Blastomyces dermatitidis is a dimorphic fungal pathogen that primarily causes blastomycosis in the midwestern and northern United States and Canada. While the genes controlling sexual development have been known for a long time, the genes controlling sexual reproduction of B. dermatitidis (teleomorph, Ajellomyces dermatitidis) are unknown. We identified the mating-type (MAT) locus in the B. dermatitidis genome by comparative genomic approaches. The B. dermatitidis MAT locus resembles those of other dimorphic fungi, containing either an alpha-box (MAT1-1) or an HMG domain (MAT1-2) gene linked to the APN2, SLA2, and COX13 genes. However, in some strains of B. dermatitidis, the MAT locus harbors transposable elements (TEs) that make it unusually large compared to the MAT locus of other dimorphic fungi. Based on the MAT locus sequences of B. dermatitidis, we designed specific primers for PCR determination of the mating type. Two B. dermatitidis isolates of opposite mating types were cocultured on mating medium. Immature sexual structures were observed starting at 3 weeks of coculture, with coiled-hyphae-containing cleistothecia developing over the next 3 to 6 weeks. Genetic recombination was detected in potential progeny by mating-type determination, PCR-restriction fragment length polymorphism (PCR-RFLP), and random amplification of polymorphic DNA (RAPD) analyses, suggesting that a meiotic sexual cycle might have been completed. The F1 progeny were sexually fertile when tested with strains of the opposite mating type. Our studies provide a model for the evolution of the MAT locus in the dimorphic and closely related fungi and open the door to classic genetic analysis and studies on the possible roles of mating and mating type in infection and virulence.

Blastomycosis

Blastomycosis is a systemic mycosis with a high prevalence in the Midwest of the United States. The fungus inhabits soil, and human infection occurs through inhalation. Its asexual phase is called Blastomyces dermatitidis and its sexual phase, Ajellomyces dermatitidis. It is more common in men. Signs and symptoms are usually severe, starting with an infection resembling pneumonia that later disseminates to the skin, bones, and central nervous system. Infection in dogs is common in endemic areas. The diagnosis can be achieved by identifying the organism with direct microscopy, culture, histopathology, serologic tests, and molecular techniques, although these are still in trial phase. The treatments of choice are azoles (itraconazole, fluconazole, and posaconazole), and in severe cases, amphotericin B.

Probable Blastomyces dermatitidis Infection in a Young Rat

A 21-week-old male untreated control SHR/NCrlNarl rat was found dead during an experiment. Grossly, pulmonary lesions were characterized by multifocal to coalescing firm gray-white nodules randomly scattered on the surface. Microscopically, bronchopneumonia was found with pyogranulomas containing neutrophils, macrophages, and numerous thick-walled yeast cells. Yeast cells, 5 to 25 μm in diameter, with no branching of hyphae were observed by staining with hematoxylin and eosin, Diff-Quik, and periodic acid-Schiff. Furthermore, polymerase chain reaction (PCR) using panfungal and nested PCR primers were used for detection of Blastomyces dermatitidis DNA in the lung tissue. After sequencing and matching with DNA sequences in the GenBank, the sample showed a similarity of 94.6% and 97% to Ajellomyces dermatitidis (B. dermatitidis), respectively. On the basis of these results, probable pulmonary blastomycosis was diagnosed. The origin of the infection in the colony rat is undetermined.

What is your diagnosis? Subcutaneous mass fluid from a febrile dog

A 10-month-old spayed female Doberman Pinscher was presented for lameness. On physical examination, the dog was lethargic and febrile and had a 2-cm raised subcutaneous mass at the base of the left ear. Fluid from the mass was drained. Direct smears of the fluid, stained with modified Wright's and new methylene blue, were highly cellular and contained large numbers of degenerate neutrophils with moderate numbers of macrophages. Large numbers of round yeast organisms, 8-20 mum in diameter, were observed extracellularly. The organisms had a thick blue wall and granular internal contents and broad-based budding was seen frequently. Branching hyphae or pseudohyphae, with parallel sides and 2-4 mum in diameter, appeared to extend from the surface of the yeast. The morphology of the yeast organisms was consistent with Blastomyces dermatitidis, with atypical hyphae formation. Culture results were not definitive because it was not possible to induce transition from the mycelial to the yeast form at 37 degrees C and because the morphology of the mycelial form of B. dermatitidis could not be differentiated from that of Emmonsia parvae. The organism was confirmed as Ajellomyces dermatitidis (the mycelial form of B. dermatitidis) using 18S ribosome RNA gene sequencing and comparison with an available databank. The mycelial form of B. dermatitidis is rarely found in the tissue of dogs, and may have been induced in this case by low environmental temperatures and the time delay between sample collection and slide preparation.

Molecular genetic variation in Emmonsia crescens and Emmonsia parva, etiologic agents of adiaspiromycosis, and their phylogenetic relationship to Blastomyces dermatitidis (Ajellomyces dermatitidis) and other systemic fungal pathogens.

Emmonsia crescens, an agent of adiaspiromycosis, Blastomyces dermatitidis, the agent of blastomycosis, and Histoplasma capsulatum, the agent of histoplasmosis, are known to form meiotic (sexual) stages in the ascomycete genus Ajellomyces (Onygenaceae, Onygenales), but no sexual stage is known for E. parva, the type species of the genus Emmonsia. To evaluate relationships among members of the putative Ajellomyces clade, large-subunit ribosomal and internal transcribed spacer region DNA sequences were determined from PCR-amplified DNA fragments. Sequences were analyzed phylogenetically to evaluate the genetic variation within the genus Emmonsia and evolutionary relationships to other taxa. E. crescens and E. parva are distinct species. E. crescens isolates are placed into two groups that correlate with their continents of origin. Considerable variation occurred among isolates previously classified as E. parva. Most isolates are placed into two closely related groups, but the remaining isolates, including some from human sources, are phylogenetically distinct and represent undescribed species. Strains of B. dermatitidis are a sister species of E. parva. Paracoccidioides brasiliensis and Histoplasma capsulatum are ancestral to most Emmonsia isolates, and P. brasiliensis, which has no known teleomorph, falls within the Ajellomyces clade.

Treatment and experimental therapeutics of blastomycosis

In 1894, blastomycosis (Gilchrist’s disease) was first described by Gilchrist as a protozoan infection [1,2]. However, in 1896, Gilchrist and Stokes [3] identified the pathogen as a fungus and named it, Blastomyces dermatitidis. Two years later, the disease was successfully transferred by inoculation of material from a human lesion to a dog [4]. Later, other cases of blastomycosis were reported by physicians, mainly from the Chicago area, and their keen interest in this mycosis led to its recognition at one time as the ‘Chicago disease’. Blastomycosis was originally believed to be endemic to North and Central America (U.S., Canada, Mexico, and Central America) until 1952, when a case was reported from Tunisia in which the pathogen was identified as Scopulariopsis americana, a synonym of B. dermatitidis [5,6]. Later, other cases of blastomycosis in Africa, South America and Asia followed [7-131. In 1968, McDonough and Lewis [14] discovered the ascomycetous state (Ajellomyces dermatitidis) of B. dermatitidis and provided a method for testing further the identity of fungi from North American and African blastomycosis. Recently, some of the clinical features, diagnosis and treatment of blastomycosis in Africa, have been reviewed [15]. The exact incidence and epidemiology of blastomycosis is not well understood mainly because of the lack of a reliable immunologic marker from previous infections. Blastomyces (Ajellomyces) dermatitidis is a dimorphic fungus that grows in mammalian tissues as budding cells (yeast phase), and in cultures as a dry, white mold (mycelial form) bearing spherical or ovoid conidia (arthrospores), 2 to 10 pm in diameter [16-l 81. The arthrospores represent the infectious particles of the parasite [19]. Inhalation of these conidia results in primary infection in the

Enzymatic activity profiling as a potential biotyping method for Ajellomyces dermatitidis

Enzyme profiling was investigated as a means of recognizing biotypes and individual strains of Ajellomyces dermatitidis (anamorph, Blastomyces dermatitidis). Eighteen North American and 2 African representatives were tested with the Yeast-IDENT enzymatic activity profiling system (Analytab Products, Plainview, N.Y.). Significant variation was found between isolates, particularly in beta-galactosaminidase activity.

Survival and growth of Ajellomyces (Blastomyces) dermatitidis on oak leaves coated with saliva

Yeast-form cells of Ajellomyces dermatitidis transferred to unsterilized and sterilized oak leaves in a humidity chamber failed to grow and produce mycelium. Transfers of these cells to Mycobiotic agar resulted in the growth of A. dermatitidis from all 5 autoclaved and 4 of the 11 unsterilized leaves. Soaking oak leaves with human airways secretions or saliva and inoculating them with yeast-form cells, and pouring sterile H2O on the leaves 10 days to 2 months later, permitted growth on 11 out of 36 leaves. It was concluded that these two natural substances, airways secretions and saliva, inhibited bacteria and furnished nutriment to A. dermatitidis.

Dual infection by "+" and "-" mating types of Ajellomyces (Blastomyces) dermatitidis.

A culture of Blastomyces dermatitidis isolated via bronchial washing from a patient with blastomycosis was studied intensively. The primary isolate was shown to have contained both the "+" and "-" mating type determiners of Ajellomyces dermatitidis. This was evidenced by the observations that both a first and a second subculture, when paired with itself produced fertile ascocarps typical of A. dermatitidis. Both these subcultures determined the production of ascospores when paired individually with each of six testers, these testers not producing fertile fruiting bodies when selfed. Isolation by micromanipulation of "+" and "-" ascospores from ascocarps produced by selfing a subculture established the mixed ("+", "-") nature of the clinical isolate and of the infection. The mixed infection was considered to have resulted from inhalation of both the "+" and "-" ascospores or conidia of A. dermatitidis or from the inhalation of one spore that contained both the "+" and "-" mating type determiners.

Blastomycos1s: Specificity of antigens reflecting the mating types of Ajellomyces dermatitidis

In a study of sera from patients with proven or suspected blastomycosis, positive immunodiffusion tests were obtained in all active cases when fresh sera were tested with a cell sap (CS) antigen. False negatives occurred on occasion when an ethanol precipitate (EPF) antigen was used alone. No false positives were found. The CS antigen from the (+) mating type had in common two lines of identity with the (CS) antigen of the (-) type. In addition, other lines were present when the patient was infected with the same mating type as was used for the preparation of the antigen. No differences in the electrophoretic patterns of the enzymes leucine amino peptidase or phosphatase were noted when preparations from the two mating types were compared. However, a distinct pattern was noted when the alpha esterases of the (+) and (-) mating types were examined. Specific alpha esterase antibodies were present in patients' sera.

Observations of a naturally-occurring lattice array of ribosomes in ultrathin section of Ajellomyces dermatitidis.

A highly organized crystalline lattice array believed to represent an intact naturally-occurring polyrisobome was observed in ultrathin section of a hyphal cell ofAjellomyces dermatitidis. This remarkable structure was composed of approximately 120 ribosomal particles in a high degree of 3-dimensional order. Connecting fibers thought to be informational ribonucleic acid were clearly demonstrated between the adjacent ribosomal particles of the lattice array. Subsequent attempts to induce ordered arrays of ribosomes in the hyphae ofA. dermatitidis by pre-cooling and treatment with physiological concentrations of vinblastine sulfate were not successful. It was concluded that the demonstration of naturally-occurring polyribosomes in this fungal organism by conventional techniques of electron microscopy was entirely fortuitous, and that such observation in ultrathin sections is an exceedingly rare event. Further, we believe this report to be the first demonstration by electron microscopy of an intact naturally-occurring polyribosome from fungal tissue.

Ultrastructural Comparison of Coils and Ascospores of Emmonsiella Capsulata and Ajellomyces Dermatitidis

Ultrastructural Comparison of Coils and Ascospores of Emmonsiella Capsulata and Ajellomyces Dermatitidis

Deoxyribonucleic acid base composition of the yeastlike and mycelial phases of Histoplasma capsulatum and Blastomyces dermatitidis.

The base composition in moles percent guanine plus cytosine (%GC) of both nuclear and mitochondrial deoxyribonucleic acid (DNA) isolated from the yeastlike and mycelial phases of the dimorphic fungal pathogens Histoplasma capsulatum and Blastomyces dermatitidis was determined by techniques of thermal denaturation and CsCl buoyant density gradient equilibrium centrifugation. The mean observed values for GC content of nuclear DNA from H. capsulatum and B. dermatitidis were 47.3 and 48.2%, respectively. What is speculated to be mitochondrial DNA was found to be 34.0% for H. capsulatum and 34.3% for B. dermatitidis. Thermal denaturation curves for Blastomyces DNA indicated a bimodality in thermal denaturation profiles, thereby suggesting a significant mitochondrial DNA contamination. Mitochondrial DNA appeared to represent a smaller percentage of the total DNA prepared from Histoplasma, and was not observed consistently to affect%GC values as determined by thermal denaturation profiles. On the basis of the now known perfect stage of B. dermatitidis (Ajellomyces dermatitidis) as a member of the family Gymnoascaceae, the close approximation of%GC content of nuclear DNA of this fungal organism with that of H. capsulatum suggests possible phylogenetic relationship. It is suggested that the just reported, but as yet unclassified, perfect stage of H. capsulatum may be found to be phylogenetically a primitive form of the Gymnoascaceae.

Blastomycosis-epidemiology and biology of its etiologic agent Ajellomyces dermatitidis.

From this review of previous publications and unpublished data it was concluded that there was little evidence that blastomycosis is contagious. Yet in spite of some reports thatB. dermatitidis has been isolated from soil no claim has been made that the ecologic niche in nature has been found. The logical assumption that soil is the natural habitat of the pathogen has been made less certain by demonstrations that this fungus, unlikeHistoplasma capsulatum, soon disappears from natural soils into which it is introduced. Furthermore,B. dermatitidis is lysed by microorganisms obtained from such soils. Therefore it was considered to be questionable how much epidemiologic significance is to be applied to chance contaminations of soil by animals with blastomycosis. Only one species ofAjellomyces dermatitidis, the heterothallic, perfect stage ofB. dermatitidis, may exist in the United States of America. Evidence for this was obtained through pairing cultures isolated in that country since ascospores were produced when an isolate from a dog was paired with an isolate from a person and fertile cleistothecia were produced by pairing isolates from widely distributed geographic regions. Individual pairings of 6 African isolates with testorB. dermatitidis strains from the U.S.A. resulted in each case in production of ascocarps. However, unquestioned ascospore production has not been obtained, although a few spherical cells having the dimensions of ascospores have been found in some cleistothecia. On this, and the other evidence so far obtained, it was concluded that the African isolates are closely related to the American isolates and could belong to the same species.