Proteomic analysis of the secretions of Pseudallescheria boydii, a human fungal pathogen with unknown genome.

Pseudallescheria boydii is a filamentous fungus that causes a wide array of infections that can affect practically all the organs of the human body. The treatment of pseudallescheriosis is difficult since P. boydii exhibits intrinsic resistance to the majority of antifungal drugs used in the clinic and the virulence attributes expressed by this fungus are unknown. The study of the secretion of molecules is an important approach for understanding the pathogenicity of fungi. With this task in mind, we have shown that mycelial cells of P. boydii were able to actively secrete proteins into the extracellular environment; some of them were recognized by antibodies present in the serum of a patient with pseudallescheriosis. Additionally, molecules secreted by P. boydii induced in vitro irreversible damage in pulmonary epithelial cells. Subsequently, two-dimensional gel electrophoresis combined with mass spectrometry was carried out in order to start the construction of a map of secreted proteins from P. boydii mycelial cells. The two-dimensional map showed that most of the proteins (around 100 spots) were focused at pH ranging from 4 to 7 with molecular masses ranging from 14 to >117 kDa. Fifty spots were randomly selected, of which 30 (60%) were consistently identified, while 20 (40%) spots generated peptides that showed no resemblance to any known protein from other fungi and/or MS with low quality. Notably, we identified proteins involved in metabolic pathways (energy/carbohydrate, nucleotide, and fatty acid), cell wall remodeling, RNA processing, signaling, protein degradation/nutrition, translation machinery, drug elimination and/or detoxification, protection against environmental stress, cytoskeleton/movement proteins, and immunogenic molecules. Since the genome of this fungus is not sequenced, we performed enzymatic and immunodetection assays in order to corroborate the presence of some released proteins. The identification of proteins actively secreted by P. boydii provides important new information for understanding immune modulation and provides important new perspectives on the biology of this intriguing fungus.

Molecular taxonomy and ecology of Pseudallescheria, Petriella and Scedosporium prolificans (Microascaceae) containing opportunistic agents on humans.

The main purpose of the present paper is to establish the connection between phylogenetic and morphological data and ecological features of strains of Pseudallescheria, Petriella, and Scedosporium. For the phylogenetic analysis sequences of the ITS region and the large subunit (partial sequences) of the rDNA were used. Cultural characteristics were observed on MEA 2 % and Weitzman-Silva Hutner Agar. Results showed, that three major groups could be differentiated, corresponding to Pseudallescheria, Petriella and S. prolificans. Among Petriella species only Pe. setifera is reasonably delimited. Pe. musispora was found to be synonymous with Pe. setifera. S. prolificans proved to be a homogenous species on the basis of ITS-sequences. Morphologically, Pseudallescheria and Petriella are distinguished by ostiolate vs non-ostiolate ascomata, a bipartition reflected also in ITS sequence data. We hypothesise a secondary loss of the ostiole of Pseudallescheria due to its ecological preferences. Infraspecific grouping within the highly variable species P. boydii is consistent for at least one clade in the ITS tree. The evolution of lineages with increased virulence within P. boydii is discussed.

Molecular phylogeny of the Pseudallescheria boydii species complex: proposal of two new species.

Pseudallescheria boydii (anamorph Scedosporium apiospermum) is the species responsible for human scedosporiosis, a fungal infection with a high mortality rate and which is difficult to treat. Recently, it has been demonstrated that high genetic variation exists within this species. We have performed a morphological and molecular study involving numerous strains of clinical or environmental origins and from different countries. The analysis of partial sequences of the beta-tubulin (two loci) and calmodulin genes and the internal transcribed spacer region of the rRNA gene has demonstrated that P. boydii is a species complex. The combined analysis of the sequences of the four loci of 60 strains has showed the presence of 44 haplotypes in the in group. Three species morphologically related to P. boydii sensu stricto, i.e., Pseudallescheria angusta, Pseudallescheria ellipsoidea, and Pseudallescheria fusoidea, which had previously been considered synonyms, could be differentiated genetically from P. boydii in our study. It is relevant that two of the three strains now included in P. ellipsoidea have caused invasive infections. The species Pseudallescheria minutispora and Scedosporium aurantiacum are clearly phylogenetically separated from the other species studied and are here proposed as new. Morphological features support this proposal. All the strains included in S. aurantiacum species have a clinical origin, while those included in P. minutispora are environmental. Further studies are needed to demonstrate whether all the species included in the P. boydii complex have different clinical spectra and antifungal susceptibility.

Arthritis without grains caused by Pseudallescheria boydii.

Severe infection of the knee joint by Pseudallescheria boydii in a 23-year-old female resulted in complete destruction of the cartilaginous surface. The possibility of entry of the fungus through mild abrasions of unperforated skin is discussed. The clinical appearance is compared with that of Scedosporium prolificans. Antimycotic miconazole-itraconazole treatment, applied during a period of 4 months, was successful; no side-effects were noted.

Fungal arthritis due to Pseudallescheria boydii (Scedosporium apiospermum).

Musculoskeletal infections by fungal agents are uncommon conditions. Pseudallescheria boydii is an ubiquitous saprophytic fungus frequently involved in maduromycosis but rarely in septic arthritis. We describe a case of Pseudallescheria boydii destructive suppurative arthritis and osteomyelitis of the knee in an adult Guadeloupean man. Osteoarticular infections due to Pseudallescheria boydii are reviewed.

The first case of Pseudallescheria boydii meningitis in China--electron microscopic study and antigenicity analysis of the agent.

Reported in this paper is the first case of isolation of Pseudallescheria boydii from cerebral spinal fluid of a boy with meningitis in China. Morphology and culture were observed by light microscopy, electron scanning microscopy and transmission electron microscopy. In addition, mycelium antigen prepared by SDS-PAGE was compared with that of the other strain of Scedosporium apiospermum. Both of the strains showed more than 40 peptide lines. Their molecular weight was very similar. On the CS-930 Dual-Wavelength TLC Scanner the peaks of both antigens showed to lie in nearly the same position. It could be concluded that Pseudallescheria boydii and Scedosporium apiospermum belong to the same genus, but they are of different strains and reproductive phases.