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.

A study of the growth of Pseudallescheria boydii isolates from sewage sludge and clinical sources on tributyrin, rapeseed oil, biodiesel oil and diesel oil.

The study compared the growth of Pseudallescheria boydii isolates from sewage sludge and from clinical sources on tributyrin, rapeseed oil, biodiesel oil and diesel oil agars. The isolates grew on all substrates tested. The highest growth was observed on rapeseed oil agar, while the lowest on diesel agar. On tributyrin agar, hydrolysis zones were observed around or underneath the colonies. On rapeseed oil agar, no hydrolysis zones were formed, while most isolates formed such a zone on biodiesel oil agar. Rapeseed oil and biodiesel oil stimulated the growth of P. boydii isolates, while tributyrin inhibited fungal growth. The stimulation or inhibition effect of diesel oil was dependent on the specified strain. In clinical isolates, fungal growth and activity were found to be more variable compared to sludge isolates. The data suggest that contamination of the environment with these oils could favor the growth of P. boydii. However, no association was found between the growth and oil utilization by this fungus and its pathogenicity.

Extracellular peptidase in the fungal pathogen Pseudallescheria boydii.

Pseudallescheria boydii is a ubiquitous filamentous fungus capable of causing invasive disease in humans. In the present study, using sodium dodecyl sulfate-polyacrylamide gels containing bovine serum albumin as co-polymerized substrate, we identified a 28-kDa proteolytic activity released to the extracellular environment by mycelia of P. boydii. This peptidase was detected during the growth of P. boydii in Sabouraud-dextrose medium for 13 days and reached its maximal production on day 7. The 28-kDa peptidase was active in acidic pH (5.5) and had its activity completely blocked by 1,10-phenanthroline, a potent zinc-metallopeptidase inhibitor. Two other metallopeptidase inhibitors, EDTA and EGTA, were also tested and no alterations were observed in the activity of the 28-kDa extracellular peptidase. Likewise, E-64 (a cysteine peptidase inhibitor), phenylmethylsulphonyl fluoride (a serine peptidase inhibitor), and pepstatin A (an aspartyl peptidase inhibitor) did not significantly alter the enzymatic behavior. Collectively, we described for the first time the expression of an extracellular metallopeptidase in the human opportunistic fungal pathogen P. boydii.

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.

Involvement of peptidorhamnomannan in the interaction of Pseudallescheria boydii and HEp2 cells.

Pseudallescheria boydii is an emerging fungal pathogen that has a worldwide distribution. Virulence mechanisms of P. boydii are largely unknown. We studied the interaction between P. boydii and HEp2 cells and demonstrated that conidia of P. boydii attached to, and were ingested by, HEp2 cells in a time-dependent process. After 2 h of interaction, the conidia produced a germ-tube like projection, which was able to penetrate the epithelial cell membrane. Recently, our group characterized a peptidorhamnomannan (PRM) antigen on the cell surface of P. boydii. In order to better understand the role played by this surface glycoconjugate during cell adhesion and endocytosis, inhibition assays were performed using intact PRM and anti-PRM polyclonal antibody. When HEp2 cells were pre-treated with whole PRM molecule, the adhesion and endocytic indices were, respectively, 50% and 60% lower than in non-treated epithelial cells. Moreover, when the conidial cells were pre-incubated with anti-PRM antibodies, the adherence and endocytosis processes were inhibited in a dose-dependent manner. As PRM influenced the conidia P. boydii-HEp2 cell interaction, we also performed inhibition assays in order to observe which PRM moieties could be involved in this process. Treatment of PRM with proteinase K promoted a slight inhibition of adhesion. However, the de-O-glycosylated PRM molecule as well as the monosaccharide mannose was able to efficiently inhibit the adhesion and endocytic processes. In addition, our results indicate for the first time that P. boydii PRM binds to a polypeptide of 25 kDa on the HEp2 cell surface.

Disseminated Pseudallescheria boydii (Scedosporium apiospermum) infection in a renal transplant patient.

Transplant recipients receive a number of immunosuppressive medications that result in an increased risk of infection, including infections with microbes that are normally not pathogenic. We describe a patient with end-stage renal disease who underwent kidney transplantation. Six months postoperatively, he presented with a lesion on his ankle, multiple thigh nodules, and right testicular pain. Biopsy of the ankle lesion demonstrated Pseudallescheria boydii (Scedosporium apiospermum), a common environmental fungus. Following orchiectomy, multiple fungal elements were found that were initially described as Aspergillus species, but later identified as P. boydii. In addition, multiple brain abscesses were found on magnetic resonance imaging. Despite treatment with multiple antifungal medications, the patient died of cardiac dysrhythmia. Current diagnostic and therapeutic alternatives for P. boydii are reviewed.

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.

Nasal granuloma caused by Scedosporium apiospermum in a dog.

A 10-month-old male American Staffordshire terrier was presented to the Autonomous University of Barcelona Veterinary Teaching Hospital because of a 6-month history of a mucopurulent bilateral nasal discharge. The dog had not responded to antibiotics. A follow-up X ray revealed a mixed pattern of osteolysis and increased radiodensity confined to the nasal cavity. Histologic sections of the biopsy specimens revealed the presence of granules containing numerous septate hyphae that were hyaline to pale brown and smooth, one-celled, subspherical-to-elongate conidia that were hyaline to brownish green, and bacteria. Cultures yielded numerous colonies belonging to Scedosporium apiospermum. Susceptibility tests were performed on the isolated strain. The isolate was sensitive to ketoconazole, intermediate to clotrimazole, and resistant to amphotericin B, 5-fluorocytosine, fluconazole, and itraconazole. The dog was treated with oral ketoconazole. During the treatment a general improvement in the lesions was observed. To our knowledge, S. apiospermum has not been implicated previously as an etiologic agent of nasal disease in dogs. This report provides its first description as such.

In vitro interactions between Blastomyces dermatitidis and other zoopathogenic fungi.

The results of in vitro interactions between colonies of Blastomyces dermatitidis and six other zoopathogenic fungi are reported. The interactions were found to range from neutral with Histoplasma capsulatum and Candida albicans to strongly antagonistic with Microsporum gypseum, Pseudallescheria boydii, and Sporothrix schenckii, and including lysis by Cryptococcus neoformans. These observations suggest that interactions between zoopathogenic fungi may be one of the biotic factors likely to influence the occurrence of B. dermatitidis in natural systems.

Adenylate energy charge and the steroid 11 alfa-hydroxylase activity in Monosporium olivaceum during growth and starvation.

During growth of Monosporium olivaceum its energy charge, E.C., (i.e. the adenylates ratio ATP + 0.5 ADP/ATP + ADP + AMP) increased from an initial value of 0.59 up to 0.85 after 25 hr of growth and then decreased to 0.51. The increase of energy charge was followed by the decrease of the activity of the 11 alpha-hydroxylase of cortexolone. This occured very clearly in the starved mycelium. Highest hydroxylation activity was observed when the lowest E.C. level (0.39-0.33) was reached.

Changes in the cellular content of the pool constituents of Monosporium olivaceum -- a steroid hydroxylating mould.

Changes of the metabolic pool constitutents of Monosporium olivaceum -- a mould capable of steroid hydroxylation were examined. The experiments were carried during growth and starvation of the microorganism. The highest activity of the 11alpha-hydroxylase was observed in the mycelium which contained the lowest level of free amino acids, glucose, and mannitol. It is suggested that the inhibition of biosynthetic processes and the decrease of the respiration rate, the activity of the NAD(P)H regenerating systems maintained, provide the optimal physiological conditions for the activity of the steroid hydroxylases.