Cell wall modifications during conidial maturation of the human pathogenic fungus Pseudallescheria boydii.

Progress in extending the life expectancy of cystic fibrosis (CF) patients remains jeopardized by the increasing incidence of fungal respiratory infections. Pseudallescheria boydii (P. boydii), an emerging pathogen of humans, is a filamentous fungus frequently isolated from the respiratory secretions of CF patients. It is commonly believed that infection by this fungus occurs through inhalation of airborne conidia, but the mechanisms allowing the adherence of Pseudallescheria to the host epithelial cells and its escape from the host immune defenses remain largely unknown. Given that the cell wall orchestrates all these processes, we were interested in studying its dynamic changes in conidia as function of the age of cultures. We found that the surface hydrophobicity and electronegative charge of conidia increased with the age of culture. Melanin that can influence the cell surface properties, was extracted from conidia and estimated using UV-visible spectrophotometry. Cells were also directly examined and compared using electron paramagnetic resonance (EPR) that determines the production of free radicals. Consistent with the increased amount of melanin, the EPR signal intensity decreased suggesting polymerization of melanin. These results were confirmed by flow cytometry after studying the effect of melanin polymerization on the surface accessibility of mannose-containing glycoconjugates to fluorescent concanavalin A. In the absence of melanin, conidia showed a marked increase in fluorescence intensity as the age of culture increased. Using atomic force microscopy, we were unable to find rodlet-forming hydrophobins, molecules that can also affect conidial surface properties. In conclusion, the changes in surface properties and biochemical composition of the conidial wall with the age of culture highlight the process of conidial maturation. Mannose-containing glycoconjugates that are involved in immune recognition, are progressively masked by polymerization of melanin, an antioxidant that is commonly thought to allow fungal escape from the host immune defenses.

El complejo Pseudallescheria/Scedosporium y taxas afines en la rutina del laboratorio micológico clínico / The Pseudallescheria/Scedoporium complex and related taxa in the routine of clinic mycological laboratory

El complejo Pseudallescheria/Scedosporium y taxas relacionados, pertenecientes a un grupo de microhongos con conidios viscosos e integrantes del orden Microascales, se presentan en diversos ambientes comunes asociados a las actividades humanas, ya sea en el suelo como en aguas contaminadas. Actualmente se consideran entre los mayores grupos de hongos filamentosos oportunistas causante de infecciones cutáneas y profundas en el hombre y otros mamíferos. El objetivo principal de este trabajo, consiste en reunir los datos primarios morfofisiológicos más relevantes, útiles para el micólogo médico en el laboratorio, con el aporte adicional de algunos aspectos, biológicos, ecológicos, taxonómicos y moleculares complementarios descritos en la literatura moderna.

The Pseudallescheria/Scedosporium complex and their related taxa are a group of fungus that conidia are presents in viscous mass and belong to the order Microascales. They are in several common enviroment related to human activities either in soil as in contaminated water. Nowadays they are considerated one of the most opportunistic group of filamentous fungus that may cause superficial and deep skin mycoses infections in man and other mammalian. The aim of this work is to gather the primary relevant morphophysiological aspects, usefull to the medical mycologist in the laboratory, plus the contribution of some of biological, ecological, taxonomical and moleculars complementary aspects that are describe in modern literature.

Severe prosthetic joint infection in an immunocompetent male patient due to a therapy refractory Pseudallescheria apiosperma.

Prosthetic joint infections (PJI) are rarely due to fungal agents and if so they are mainly caused by Candida strains. This case represents a PJI caused by a multi-drug resistant Pseudallescheria apiosperma, with poor in vivo response to itraconazole and voriconazole. This case differs also by the way of infection, since the joint infection did not follow a penetrating trauma. In the majority of cases, Scedosporium extremity infections remain local in immunocompetent individuals. We report a persistent joint infection with multiple therapeutic failures, and subsequent amputation of the left leg. Detailed clinical data, patient history, treatment regime and outcome of a very long-lasting (>4 years) P. apiosperma prosthetic knee infection in an immunocompetent, 61-year-old male patient are presented with this case. The patient was finally cured by the combination of multiple and extensive surgical interventions and prolonged antifungal combination therapy with voriconazole and terbinafine.

Disseminated Scedosporium/Pseudallescheria infection after double-lung transplantation in patients with cystic fibrosis.

We report a case of disseminated Scedosporium/Pseudallescheria infection due to Pseudallescheria boydii sensu stricto after lung transplantation in a patient with cystic fibrosis. Dissemination occurred under voriconazole. Despite surgery and combination therapy with voriconazole, caspofungin, and terbinafine, the patient died 8 months after transplantation. Previously reported cases are reviewed.

Production of a fungistatic substance by Pseudallescheria boydii isolated from soil amended with vegetable tissues and its significance.

Four fungal isolates that were able to use vegetable tissues for multiplication in soil were isolated and identified as Pseudallescheria boydii based on morphological characteristics and ITS sequence similarity. When grown in broth prepared from the same vegetable tissues used in soil amendment, all these isolates of P. boydii produced a substance capable of reducing the disease incidence of black leaf spot of spoon cabbage caused by Alternaria brassicicola and inhibiting the germination of A. brassicicola conidia. The substance, which was fungistatic, was very stable under high temperature and high or low pH value. It was soluble in polar solvents and insoluble in non-polar solvents. Molecular weight estimation and ion exchange ability tests suggest that the fungistatic compound has a molecular weight between 500 and 1,000 and has no charge on its molecule. Results from this study suggest the possession of a strong competitive saprophytic ability by P. boydii, which in turn may explain the widespread occurrence of this human pathogen in soil. Production of a fungistatic substance when P. boydii was grown in broth prepared from vegetable tissues suggests the importance of antibiotic production in its competitive saprophytic colonization of organic matters in soil.

Heterothallism in Scedosporium apiospermum and description of its teleomorph Pseudallescheria apiosperma sp. nov.

Scedosporium apiospermum has traditionally been thought of as the anamorph of Pseudallescheria boydii (Microascaceae, Ascomycota), but recent molecular studies has demonstrated that they are different species. Since a teleomorph was not observed among isolates recently identified as S. apiospermum, we investigated whether this species could be heterothallic. In this study, 15 isolates of S. apiospermum were paired in all possible combinations, including self-pairings. Several combinations produced fertile ascomata typical of the genus Pseudallescheria, while all isolates were self-sterile. The isolates were grouped into two different mating types. Crosses among F1 progeny ascospores demonstrated a bi-allelic heterothallic mating system. The new species Pseudallescheria apiosperma, teleomorph of S. apiospermum, is proposed and described.

Ecology of Pseudallescheria and Scedosporium species in human-dominated and natural environments and their distribution in clinical samples.

This study aims to determine the occurrence of Pseudallescheria and Scedosporium species in natural and human-dominated environments. Habitats (136 sampling sites) in a transect with increasing human impact were investigated (natural areas, agricultural soils, urban playgrounds, industrial areas). Physico-chemical parameters were measured to characterize the different areas included in this investigation. Fungal identification was performed by morphology and sequence data analysis. Comparative description of virulence was largely based on the database of the ECMM/ISHAM Working Group on Pseudallescheria/Scedosporium Infections. Pseudallescheria and Scedosporium species were most abundant in industrial areas, followed by urban playgrounds and agricultural areas. None of the species were isolated from natural habitats. The abundance of Pseudallescheria and Scedosporium species could be correlated with increasing nitrogen concentrations (P<0.01) and decreasing pH (P<0.05) within a pH range of 6.1-7.5. In general, frequency of the different Pseudallescheria and Scedosporium species in the environment is strongly enhanced by human activities, and largely differs from species distribution in clinical settings, suggesting that these species have different degrees of virulence. Pseudallescheria boydii is relatively frequently found as agent of human disease, while Scedosporium dehoogii is found almost exclusively in the environment. Scedosporium apiospermum is responsible for the majority of infections and is found at comparable frequency in the environment; S. aurantiacum and P. minutispora showed similar spectra, but at much lower frequencies.

Molecular and phenotypic data supporting distinct species statuses for Scedosporium apiospermum and Pseudallescheria boydii and the proposed new species Scedosporium dehoogii.

Based on the morphological, physiologic, and molecular (beta-tubulin gene) study of 141 isolates of the Pseudallescheria boydii species complex (including several synonyms) and relatives, the new species Scedosporium dehoogii is proposed. Scedosporium apiospermum and P. boydii are considered two different species and the new name Scedosporium boydii is proposed for the anamorph of the latter species. A summary of the key morphological and physiological features for distinguishing the species of Pseudallescheria/Scedosporium is provided.

Characterization of glucosylceramides in Pseudallescheria boydii and their involvement in fungal differentiation.

Pseudallescheria boydii is a fungal pathogen that causes disease in immunocompromised patients. Ceramide monohexosides (CMHs) were purified from lipidic extracts of this fungus, showing that, as described for several other species, P. boydii synthesizes glucosylceramides as major neutral glycosphingolipids. CMHs from P. boydii were analyzed by high-performance thin-layer chromatography, gas chromatography coupled to mass spectrometry, fast atom bombardment-mass spectrometry, and nuclear magnetic resonance. These combination of techniques allowed the identification of CMHs from P. boydii as molecules containing a glucose residue attached to 9-methyl-4,8-sphingadienine in amidic linkage to 2-hydroxyoctadecanoic or 2-hydroxyhexadecanoic acids. Antibodies from a rabbit infected with P. boydii recognized CMHs from this fungus. Antibodies to CMH were purified from serum and used in indirect immunofluorescence, which revealed that CMHs are detectable on the surface of mycelial and pseudohyphal but not conidial forms of P. boydii, suggesting a differential expression of glucosylceramides according with morphological phase. We also investigated the influence of antibodies to CMH on growth and germ tube formation in P. boydii. Cultures that were supplemented with these antibodies failed to form mycelium, but the latter was not affected once formed. Similar experiments were performed to evaluate whether antibodies to CMH would influence germ tube formation in Candida albicans, a fungal pathogen that synthesizes glucosylceramide and uses differentiation as a virulence factor. Addition of antiglucosylceramide antibodies to cultures of C. albicans clearly inhibited the generation of germ tubes. These results indicated that fungal CMHs might be involved in the differentiation and, consequently, play a role on the infectivity of fungal cells.

Pseudallescheria: an underdiagnosed fungus?

Pseudallescheria has been identified as one of the "clinically significant emerging mycoses" but has received little attention in the cytology literature. Recognition of this fungus is of particular importance clinically, because unlike most other fungi (including Aspergillus, with which it is most frequently confused), Pseudallescheria is not effectively treated with amphotericin B, the most frequently and often the only antifungal agent administered. Features helpful in the diagnosis of Pseudallescheria in cytologic material are presented.

Degradation of hydrocarbons in crude oil by the ascomycete Pseudallescheria boydii (Microascaceae).

Four unique strains of Pseudallescheria boydii were isolated from oil-soaked soils in British Columbia and Alberta and compared to strains from cattle dung and raw sewage. Considerable variability in morphology, colony appearance, colony diameter, and temperature tolerance occurred among the strains. They also varied in the sporogenous states produced in culture; all strains had a Scedosporium anamorph and either the Graphium anamorph or cleistothecial teleomorph. Conspecificity of the six isolates was inferred from their morphology and supported by restriction fragment length polymorphism profiles of the internally transcribed spacer region of rDNA and comparing these to Petriella sordida, a similar taxon in the Microascaceae. Three of the strains isolated from oil-contaminated soil and the strain from sewage were tested for their ability to utilize hydrocarbons by incubation with Prudhoe Bay Crude oil as the sole carbon source. Gas chromatographic analysis of the residual oil revealed that the strains isolated from oil-contaminated soil degraded the linear aliphatics. The strain from sewage, previously shown by others to utilize the volatile n-alkanes (i.e., ethane, propane, and butane), did not utilize the liquid saturate compounds. None of the strains was observed to degrade compounds in the aromatic fraction. Pseudallescheria boydii may be an important agent for in situ bioremediation of saturates in oil-contaminated sites.

Fungal endophthalmitis. An experimental study with a review of 17 human ocular cases.

Pseudallescheria boydii is an opportunistic fungus that is histologically indistinguishable from Aspergillus fumigatus. Pseudallescheria boydii has been reported to cause endophthalmitis, orbital cellulitis, and corneal ulceration and is, thus, important to the ophthalmologist. A clinical review of 17 patients with P boydii ophthalmic infections is presented. In addition, animal models of endophthalmitides caused by A fumigatus and P boydii were created and compared. Dutch-Belted rabbits used for the experimental models were immunosuppressed with methylprednisolone acetate (Depomedrol). Exogenous and endogenous models of P boydii endophthalmitis were created in immunocompetent and immunosuppressed animals. An exogenous model of A fumigatus endophthalmitis was created in immunocompetent and immunosuppressed animals for comparison. The clinical and histopathologic features of A fumigatus and P boydii endophthalmitis are indistinguishable in the immunocompetent and immunosuppressed exogenously infected rabbits. Endogenous P boydii endophthalmitis has a similar fundus appearance to that caused by Nocardia asteroides.