Toll-like receptors (TLR2 and TLR4) recognize polysaccharides of Pseudallescheria boydii cell wall.

Pseudallescheria boydii is an opportunistic fungus widespread in the environment, and has recently emerged as an agent of localized as well as disseminated infections in both immunocompromised and immunocompetent hosts. The host response to fungi is in part dependent on the activation of evolutionary conserved receptors including Toll-like receptors and phagocytic receptors. This review will discuss the isolation and structural characterization of α-glucans and rhamnomannans from P. boydii cell wall and their roles in the induction of innate immune response.

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.

TLR4 recognizes Pseudallescheria boydii conidia and purified rhamnomannans.

Pseudallescheria boydii (Scedosporium apiospermum) is a saprophytic fungus widespread in the environment, and has recently emerged as an agent of localized as well as disseminated infections, particularly mycetoma, in immunocompromised and immunocompetent hosts. We have previously shown that highly purified α-glucan from P. boydii activates macrophages through Toll-like receptor TLR2, however, the mechanism of P. boydii recognition by macrophage is largely unknown. In this work, we investigated the role of innate immune receptors in the recognition of P. boydii. Macrophages responded to P. boydii conidia and hyphae with secretion of proinflammatory cytokines. The activation of macrophages by P. boydii conidia required functional MyD88, TLR4, and CD14, whereas stimulation by hyphae was independent of TLR4 and TLR2 signaling. Removal of peptidorhamnomannans from P. boydii conidia abolished induction of cytokines by macrophages. A fraction highly enriched in rhamnomannans was obtained and characterized by NMR, high performance TLC, and GC-MS. Preparation of rhamnomannans derived from P. boydii triggered cytokine release by macrophages, as well as MAPKs phosphorylation and IκBα degradation. Cytokine release induced by P. boydii-derived rhamnomannans was dependent on TLR4 recognition and required the presence of non-reducing end units of rhamnose of the rhamnomannan, but not O-linked oligosaccharides from the peptidorhamnomannan. These results imply that TLR4 recognizes P. boydii conidia and this recognition is at least in part due to rhamnomannans expressed on the surface of P. boydii.

Tracking the emerging human pathogen Pseudallescheria boydii by using highly specific monoclonal antibodies.

Pseudallescheria boydii has long been known to cause white grain mycetoma in immunocompetent humans, but it has recently emerged as an opportunistic pathogen of humans, causing potentially fatal invasive infections in immunocompromised individuals and evacuees of natural disasters, such as tsunamis and hurricanes. The diagnosis of P. boydii is problematic since it exhibits morphological characteristics similar to those of other hyaline fungi that cause infectious diseases, such as Aspergillus fumigatus and Scedosporium prolificans. This paper describes the development of immunoglobulin M (IgM) and IgG1 kappa-light chain monoclonal antibodies (MAbs) specific to P. boydii and certain closely related fungi. The MAbs bind to an immunodominant carbohydrate epitope on an extracellular 120-kDa antigen present in the spore and hyphal cell walls of P. boydii and Scedosporium apiospermum. The MAbs do not react with S. prolificans, Scedosporium dehoogii, or a large number of clinically relevant fungi, including A. fumigatus, Candida albicans, Cryptococcus neoformans, Fusarium solani, and Rhizopus oryzae. The MAbs were used in immunofluorescence and double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) to accurately differentiate P. boydii from other infectious fungi and to track the pathogen in environmental samples. Specificity of the DAS-ELISA was confirmed by sequencing of the internally transcribed spacer 1 (ITS1)-5.8S-ITS2 rRNA-encoding regions of environmental isolates.

Subcutaneous infection with Pseudallescheria boydii in an immunocompromised patient.

With the broad employment of immunosuppressive therapy, the incidence of Pseudallescheria boydii infections is rising. We report a first case of the localized subcutaneous P. boydii infection in a patient with microscopic polyangiitis. Favorable outcome related to the treatment with voriconazole adds to the growing body of evidence supporting the use of this particular agent in P. boydii infections.

Structures of the O-linked oligosaccharides of a complex glycoconjugate from Pseudallescheria boydii.

Nonreducing O-linked oligosaccharides were obtained from the peptidorhamnomannan of mycelia of Pseudallescheria boydii by alkaline beta-elimination under reducing conditions. They were separated by gel filtration chromatography to give three oligosaccharide fractions. The major oligosaccharide from fraction 1 was characterized by a combination of techniques including electrospray ionization quadrupole time-of-flight tandem mass spectrometry (ESI MS/MS), matrix-assisted laser desorption ionization mass spectrometry (MALDI MS), nuclear magnetic resonance (NMR), and methylation gas-liquid chromatography-mass spectrometry (GC-MS) analysis. It was branched, with a principal chain of alpha-Rhap-(1 --> 3)-alpha-Rhap-(1 --> 3)-alpha-Manp-(1 --> 2)-Man-ol substituted at O-6 of mannitol with an alpha-Glcp-(1 --> 4)-beta-Galp group. Species containing one and two additional alpha-Glcp-(1 --> 4) substituents in the rhamnose branch were also present. The major component of fraction 2 was a substructure of oligosaccharide-1, lacking a hexose from the Glc-Gal branch. Fraction 3 contained a mixture of smaller, unbranched, oligosaccharides. In hapten inhibition tests, fractions 1 and 2 blocked the reaction between peptidorhamnomannan (PRM) and rabbit anti-P. boydii mycelium hyperimmune serum by approximately 75%, whereas fraction 3 inhibited by approximately 55%.

A peptidorhamnomannan from the mycelium of Pseudallescheria boydii is a potential diagnostic antigen of this emerging human pathogen.

The ascomycete Pseudallescheria boydii is an emerging human pathogen frequently found in soil and polluted water. A peptidopolysaccharide antigen has been isolated from mycelial forms of P. boydii, and characterized using chemical and immunological methods. Monosaccharide composition, methylation analysis, and (1)H- and (13)C-NMR spectra indicated the presence of a rhamnomannan with a structure distinct from those of similar components isolated from other fungi, containing Rhap(1-->3)Rhap epitopes on side chains which may be linked (1-->3) to (1-->6)-linked mannose. The peptidorhamnomannan from P. boydii reacted poorly with an antiserum raised against whole cells of Sporothrix schenckii and strongly with one against P. boydii hyphae. These characteristics and immunological differences suggest that this major rhamnose-containing antigen of P. boydii may be useful for the specific diagnosis of infections attributable to this fungus.

Clinical significance of Scedosporium apiospermum in patients with cystic fibrosis.

The incidence of airway colonization by Scedosporium apiospermum and of related sensitization was investigated prospectively in 128 patients with cystic fibrosis over a 5-year period, and results were compared with clinical data. Scedosporium apio-spermum, recovered from sputum samples in 11 of 128 (8.6%) patients, was the most frequent filamentous fungus after Aspergillus fumigatus. Counterimmuno-electrophoresis, used to detect scedosporiosis serologically, was positive in 27 of 128 (21.1%) patients. The discrepancy between the mycological and serological results may be related to immune cross-reactions between Scedosporium apiospermum and Aspergillus fumigatus. However, symptoms of allergic bronchopulmonary disease were observed in two patients chronically colonized by Scedosporium apiospermum. The results clearly demonstrate that the frequency of this fungus is largely underestimated and that it may trigger an inflammatory response, thus suggesting a pathogenic role in patients with cystic fibrosis.

Immunohistologic identification of Aspergillus spp. and other hyaline fungi by using polyclonal fluorescent antibodies.

Isolation and identification of pathogenic Aspergillus and Fusarium spp. from clinical materials provide the most accurate means for establishing a diagnosis of infections by these molds. Such efforts, however, are not always successful. Histologic diagnosis also has its limitations. In vivo the hyphae of Aspergillus and Fusarium spp. are very similar and their in situ manifestations are not pathognomonic. To improve the histologic diagnosis of infections by Aspergillus and Fusarium species, we developed polyclonal fluorescent-antibody reagents to Aspergillus fumigatus and Fusarium solani and evaluated their diagnostic utilities. Our studies revealed that A. fumigatus and F. solani share epitopes not only with one another but also with other Aspergillus and Fusarium spp. as well as with Paecilomyces lilacinus and Pseudallescheria boydii. Adsorption of the A. fumigatus conjugate with cells of Fusarium proliferatum and F. solani and F. solani antiserum with cells of Aspergillus flavus resulted in reagents that distinguished Aspergillus spp. from Fusarium spp. but that still cross-stained P. lilacinus and P. boydii. Adjunctive use of a specific P. boydii conjugate enabled the identification of Aspergillus spp., Fusarium spp., P. lilacinus, and P. boydii in formalin-fixed tissue sections from 19 humans with culture-proven cases of mycotic infection.

Allergic bronchopulmonary mycosis caused by Pseudallescheria boydii.

Two cases of allergic bronchopulmonary pseudallescheriosis (ABPP) are described. These are the first cases of this allergic bronchopulmonary mycosis (ABPM) reported in which the clinical and serologic criteria are described. The first case was in a patient with mild asthma, and it resolved spontaneously after expectoration of a mucous plug. The second was in a patient with recurrent allergic bronchopulmonary aspergillosis with an exacerbation of ABPM caused by Pseudallescheria boydii. The total serum IgE, IgG, and IgE antibodies against P. boydii and the clinical picture well define ABPP. This diagnosis may be important to recognize in order to prevent a progression of the patient's lung disease.

The antigenic composition and protein profiles of eumycetoma agents.

The protein profiles of different eumycetoma agents were compared by SDS gel electrophoresis. Dendrograms confirmed the homogeneity of isolates of Pseudallescheria boydii but amongst Madurella species, particularly isolates identified as M. grisea, there were substantial differences in protein composition. However using Western blotting reference isolates of the different species showed distinct antigen patterns in response to immune rabbit sera. In particular there was little evidence of cross reactivity between M. mycetomatis and M. grisea. However this specificity was not apparent when human sera from patients with different eumycetoma infections were compared in an ELISA system using the same antigens. It is possible that the formation of a mycetoma grain may limit a patient's exposure to antigens which confer specificity, an explanation which may also account for the variability in antibody responses seen.

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.

Humoral response to experimental petriellidiosis.

The humoral antibody response elicited by experimental infection with Petriellidium boydii and Monosporium apiospermum was studied. The variables of fungus strain, route of inoculation, and concentration of inoculum were examined. Viable spores from three strains of P. boydii and from three strains of M. apiospermum were inoculated intravenously into mice. Serum specimens were tested for antibody by the microtiter indirect hemagglutination assay. At 4 weeks after infection, the percentage of positive specimens among survivortically related either to the incidence of positive specimens or to the magnitude of the humoral response. Mice inoculated by either the subcutaneous or the intramuscular route with viable or killed spores from M. apiospermum 813 developed antibody. The mean antibody titer was greater in those animals receiving viable spores, and the magnitude of the humoral response was dose dependent. Animals inoculated intravenously with comparable inocula of the same strain developed similar levels of antibody. However, cultural studies of infected mice showed chronic infection only after intravenous inoculation. The kidney was the organ most consistently involved.

[Pulmonary allescheriosis (author's transl)]. / Alleschériose pulmonaire.

In a patient properly treated for a previous cavitary tuberculosis, we had the surprise, after hemoptyses, to find a sleigh-bell shaped picture suggesting an intra-cavitary aspergilloma with a very special mycosis with Allescheria Boydii. Proof was provided by the presence of the fungus in direct examination and in culture in bronchial secretions. Surgical exeresis did not cure the patient and a secondary recurrence appeared on which antifungal drugs were ineffective. Antigens were made from the strain. They are used for immunoprecipitations tests. Only the patient's serum is positive; 82 serum test (18 concerned patients with visceral mycosis of farmer's lung disease) remained negative. Twenty two observations or pulmonary allescheriosis were recorded in the world. These figures are certainly below the truth. Three practical consequences can be drawn: --for an intra-cavitary mycetoma, with negative anti-aspergillus precipitins, such a disease must be kept in mind and the fungus looked for in bronchial secretions and specific immuno-precipitation test done; --any mycotic growth must be systematically cultured to determine the type of fungus; --for therapy, if diagnosis was made before the intervention, an antifungal treatment either local or general must be associated to the surgical treatment (contrary to aspergilloma treatment).

Serological diagnosis of petriellidosis (allescheriosis). I. Isolation and characterization of soluble antigens from Allescheria boydu and Monosporium apiospermum.

Soluble antigens in culture filtrates of three strains of Petriellidium boydii and three strains of Monosporium apiospermum were examined. Antigens were separated from concentrated crude filtrates by anion-exchange chromatography. A single major peak (Antigen 1), constituting a significant proportion of the total recoverable carbohydrate, was the only product isolated from each of four chromatographed filtrates. Depending on the fungus strain, Antigen 1 consisted of 90--96% carbohydrate, 3-4% protein, and 2-4% nucleic acid. Antigen 1 was found to consist of a population of molecules with a heterogeneous molecular size when assayed by gel filtration chromatography; however, isolated fractions of Antigen 1 proved to be immunologically identical when examined by Ouchterlony immunodiffusion. In addition, Antigen 1 from each strain was immunologically identical to similar preparations of Antigen 1 from the other five fungus strains. Chromatography of culture filtrates from two strains of M. apiospermum revealed a second peak (Antigen 2), which was found to consist of 70% carbohydrate, 16% protein, and 4% nucleic acid. Although Antigen 2 contained four times as much protein as Antigen 1, the two preparations were immunologically identical by immunodiffusion tests. Ion-exchange chromatography proved to be a useful procedure for isolating antigens of P. boydii and M. apiospermum from culture filtrates.