DIGE enables the detection of a putative serum biomarker of fungal origin in a mouse model of invasive aspergillosis.

Invasive aspergillosis (IA) is a major threat for immunocompromised patients. Diagnostic difficulties often delay specific treatment initiation, which increases mortality. Finding new biomarkers to improve and speed accurate diagnosis is thus vital. To investigate the ability of proteomic methods for discovering new biomarkers of IA, we used a DIGE approach to perform a proteomic analysis on both bronchoalveolar lavages (BAL) and sera at different time-points of infection in a mouse model of invasive pulmonary aspergillosis. Progression of the infection was monitored using a bioluminescent strain of Aspergillus fumigatus. Sera proteins were enriched using the ProteoMiner kit (Biorad). This method allowed us to identify a fungal protein, the A. fumigatus major allergen Asp f 2, in sera of mice one day after the infection. However, this fungal protein was not detected three days after the infection. Importantly, in BAL, this work provides evidence of an in vivo complement evasion mechanism through the cleavage of C3b into three fragments during aspergillosis. Finally, our results underlining the inflammatory host response to IA in both lung and blood compartments at different times of infection may provide new insights into the pathophysiology of this disease.

Interaction of Aspergillus fumigatus with the alveolar macrophage.

Aspergillus fumigatus is a human pathogen, able to cause invasive aspergillosis in immunosuppressed patients. In the immunocompetent situation inhaled conidia are easily cleared by the immune system. Knowledge of the cellular pathways involved in the innate immunity against A. fumigatus is poorly represented. Therefore, we aimed to investigate the immune response against A. fumigatus in murine alveolar macrophages in terms of MAP kinases, NF-kappaB and cytokine signalling. Our investigations revealed that in murine alveolar macrophages, MAP kinases, ERK and p38 are activated under in vitro conditions, following addition of A. fumigatus conidia. In vivo experiments, however, showed that only ERK is directly involved, because activation of p38 was negligible. Immunosuppression with corticosteroids inhibited phosphorylation of ERK and was directly accompanied with a strongly decreased level of TNF-alpha and additional cytokines. In addition, killing of A. fumigatus conidia is reduced using the ERK inhibitor. Therefore, ERK appears to be an essential MAP kinase in the defence against A. fumigatus. Activation of the transcription factor NFkappaB appeared only at late times after infection suggesting an association with the intracellular swelling of conidia.

Killing of Aspergillus fumigatus by alveolar macrophages is mediated by reactive oxidant intermediates.

Phagocytosis and mechanisms of killing of Aspergillus fumigatus conidia by murine alveolar macrophages (AM), which are the main phagocytic cells of the innate immunity of the lung, were investigated. Engulfment of conidia by murine AM lasts 2 h. Killing of A. fumigatus conidia by AM begins after 6 h of phagocytosis. Swelling of the conidia inside the AM is a prerequisite for killing of conidia. The contributions of NADPH oxidase and inducible nitric oxide synthase to the conidicidal activity of AM were studied using AM from OF1, wild-type and congenic p47phox(-/-) 129Sv, and wild-type and congenic iNOS(-/-) C57BL/6 mice. AM from p47phox(-/-) mice were unable to kill A. fumigatus conidia. Inhibitors of NADPH oxidase that decreased the production of reactive oxidant intermediates inhibited the killing of A. fumigatus without altering the phagocytosis rate. In contrast to NADPH oxidase, nitric oxide synthase does not play a role in killing of conidia. Corticosteroids did not alter the internalization of conidia by AM but did inhibit the production of reactive oxidant intermediates and the killing of A. fumigatus conidia by AM. Impairment of production of reactive oxidant intermediates by corticosteroids is responsible for the development of invasive aspergillosis in immunosuppressed mice.

Phagocytosis and intracellular fate of Aspergillus fumigatus conidia in alveolar macrophages.

Aspergillus fumigatus is the most prevalent airborne fungal pathogen responsible for fatal invasive aspergillosis in immunocompromised patients. Upon arrival in the lung alveolus, conidia of A. fumigatus are phagocytosed by alveolar macrophages, the major phagocytic cells of the lung. Engulfment and intracellular trafficking of A. fumigatus conidia in alveolar macrophages of two different origins, the murine cell line MH-S and human pulmonary alveolar macrophages, were analyzed by electron microscopy and immunofluorescence. Phagocytosis of A. fumigatus conidia required actin polymerization and phosphatidylinositol 3-kinase activity. Fusion of A. fumigatus phagosomes with early and late endosomes was shown by immunolabeling with specific markers for the transferrin receptor, early endosome antigen, and Rab7. Maturation of A. fumigatus phagolysosomes was monitored by using a fixable acidotropic probe, LysoTracker Red DND-99, and an anti-cathepsin D antibody. Bafilomycin A-induced inhibition of lysosomal acidification abolished the conidial killing by the macrophages. These data suggest that the maturation of A. fumigatus phagosomes results from fusion with the compartments of the endocytic pathway and that the killing of conidia depends on phagolysosome acidification. A model for the phagocytosis of A. fumigatus conidia by alveolar macrophages is proposed on the basis of these results.

Separation of proteases: old and new approaches.

All methods of protein separations can be applied to proteases. Some emphasis is put in this review on a powerful technique specific to proteases purification: cyclic peptide antibiotics may be seen as general affinity ligands for proteases. Also, some examples of affinity chromatography of proteases on ligands with narrower specificity are given. The special interest of hydrophobic interaction chromatography for proteases purification is discussed. The merits of immobilized dye chromatography for proteases purification and the interest in empirically screening many immobilized dyes, as well as several eluents are discussed.

Expression of PZ-peptidases by cultures of several pathogenic fungi. Purification and characterization of a collagenase from Trichophyton schoenleinii.

Peptidolytic activity was studied in the broken-cell extracts of 17 isolates of pathogenic fungi tested with phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg (PZ-PLGPA) as a substrate. All the fungi studied except Candida spp., Cryptococcus neoformans and two actinomycetes hydrolyzed the substrate and therefore contained a so-called PZ-peptidase activity. Of all the positive strains, Trichophyton schoenleinii, a pathogenic fungus showed the highest activity and was therefore chosen as a source for PZ-peptidase purification. The four chromatographic steps, a 'negative' dye column, a 'positive' dye column, hydroxyapatite Ultrogel, and modified TSK (HW 55), gave a highly purified peptidase with a 12% overall yield. Inhibitor studies suggested that the 82 000 M(r) PZ-peptidase is a metalloproteinase. Moreover it cleaved native rat type I collagen. Partial peptide sequencing showed a strong sequence homology to regions of two metalloproteinases previously identified in the yeast Saccharomyces cerevisiae and in rat.

Aspergillus fumigatus metalloproteinase that hydrolyses native collagen: purification by dye-binding chromatography.

A proteinase was purified from the human pathogenic fungus Aspergillus fumigatus. The four chromatographic steps, a "negative" dye column, a "positive" dye column, hydroxyapatite Ultrogel, and modified TSK gel (HW 55), gave a 14% overall yield. The protein migrated as a single band on SDS-PAGE and isoelectric focusing, with an M(r) of 82,000 and a pI of 5.6. Inhibitor studies suggested that the enzyme was a metalloproteinase. It hydrolyzed phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-Arg and cleaved native rat type I collagen.

Heat-shock response in Fonsecaea pedrosoi, a pathogenic fungus.

Using two-dimensional electrophoresis we have investigated the heat-shock response in a pathogenic fungus, Fonsecaea pedrosoi. Fungal cultures were transferred from 37 to 45 degrees C for either 30 or 90 min and then returned back to the initial temperature. Analysis of the total proteins resolved on two-dimensional gels indicated important changes in the accumulation of several peptides according to the duration of treatment and the temperature. The 30-min incubation at 45 degrees C resulted in the induction of several new proteins, whereas other proteins were either increased or decreased. These inductions and repressions of proteins (called heat-shock and heat-stroke proteins, respectively) were either specific to this time period or still present after a 90-min incubation. In addition, the 90-min incubation period led to the enhancement of several proteins, which were therefore called late heat-shock proteins to distinguish them from the early ones detected after 30 min. Finally, when cultures were shifted back to 37 degrees C most of the heat-shock proteins decreased or disappeared; in parallel, most of the heat-stroke proteins were reinduced at this time. These results are in good agreement with previous studies on the heat-shock response and provide additional evidence that this phenomenon is highly conserved among species.

Immunochemical characterisation of antigens and growth inhibition of Fonsecaea pedrosoi by species-specific IgG.

Antigens of Fonsecaea pedrosoi, the most common agent of chromomycosis, were characterised by immunoprecipitation with a rabbit antiserum raised against the cell-protein extract and serum from an infected patient. Thirteen antigens were commonly detected and, as some of these antigens could be iodinated, they may be present in the fungal cell wall. Purified IgG from the rabbit antiserum was shown to produce a 50-60% inhibition of fungal growth. Some of the antigens characterised may be important in relation to the stimulation of protective immunity against chromomycosis.

Polymorphism in mitochondrial DNA of several Trichophyton rubrum isolates from clinical specimens.

Mitochondrial DNA from six isolates of Trichophyton rubrum (a fungal pathogen of humans) was isolated, purified and digested by restriction endonucleases HindIII, HaeIII, AluI and EcoR1. The isolates could be classified into two groups according to their characteristic fragment patterns. Electron microscopy indicated that T. rubrum mitochondrial DNA are circular molecules of different lengths, 26.85 microns and 21.60 microns respectively, according to the group.

Comparative electrophoresis, isoelectric focusing and numerical taxonomy of some isolates of Fonsecaea pedrosoi and allied fungi.

The proteins in broken-cell extracts from eight isolates of Fonsecaea pedrosoi, the principal agent of chromomycosis, were studied and compared by electrophoresis and isoelectric focusing. A type pattern was established with 16 fractions ranging in molecular weight between 7600 and 78500 daltons and 16 fractions varying in isoelectric point between 4.95 and 7.90. A genetic distance of 0.1 found by the numerical study applied to both analyses reveals a considerable similarity among the isolates studied. This resemblance was moreover observed between F. pedrosoi and other related dematiaceous fungi.

Study of the conidial development and cleistothecium-like structure of some strains of Fonsecaea pedrosoi. Comparison with other close Dematiaeae.

The production of twenty-seven strains of Fonsecaea pedrosoi was studied. The denticulate type (asexual reproduction) showed three morphological variations: medium-size, long and sessile forms. These forms can be used to characterize the different strains isolated especially for epidemiological purposes. Only one strain (MR 1335) isolated from a chromomycosis case in Martinique showed a cleistothecium-like structure. This formation was compared to that observed in Phialophora verrucosa, another agent of chromomycosis, and to the cleistothecium of Dictyotrichiella mansonii, the perfect state of Wangiella mansonii which is a saprophyte fungus.