Isolation and immunochemical characterization of fractions from membranes of Aspergillus fumigatus with protease activity.

Two fractions exhibiting acid protease activity (AFPI and AFPII) were isolated by extraction of membrane vesicles of Aspergillus fumigatus with Triton X-100. These two fractions produced single bands in both polyacrylamide and sodium dodecyl sulfate polyacrylamide gel electrophoresis and showed apparent molecular weights of 73,000 and 43,000, respectively. Molecular weights determined by gel filtration in the absence and presence of Triton X-100 and sedimentation velocities in analytical ultracentrifugation indicated hydrophobic characteristics, since both fractions readily aggregated and complexed with Triton X-100; both exhibited elevated enzyme activities in the presence of Triton X-100. Carbohydrate content was 93% for AFPI and 85% for AFPII. The enzymatic fractions demonstrated different pH optima in the acid range as well as different temperature stabilities. Both protease fractions cross reacted in double immunodiffusion, while in crossed immunoelectrophoresis both demonstrated five precipitin peaks, each with similar patterns. AFPI demonstrated two additional precipitin peaks in crossed immunoelectrophoresis. As determined by crossed immunoaffinoelectrophoresis, the protease fractions demonstrated galactose and mannose residues. In biotin-avidin enzyme-linked immunosorbent assay both fractions reacted with allergic bronchopulmonary aspergillosis and aspergilloma sera. It can be concluded that two fractions with protease activity of A. fumigatus reported here may be of significance in Aspergillus-induced diseases.

Biochemical and immunochemical analyses of detergent solubilized antigens from membrane vesicles of Aspergillus fumigatus.

A membrane vesicle fraction isolated from exponentially growing Aspergillus fumigatus strain Ag 507 cultures was obtained by mechanical disruption of intact Aspergillus cells under specific osmotic conditions followed by a pH fractionation technique. Electron micrographs of the membrane vesicles indicated unit membrane structures free from cell wall material. High glucose-6-phosphatase and low lactate dehydrogenase activities verified the relative purity of the membrane vesicle fraction. Allergic bronchopulmonary aspergillosis (ABPA) patient and normal human sera were incubated with the membrane vesicle fraction followed by colloidal gold tagged rabbit antiserum to human IgG or IgE. Electron micrographs indicated ABPA patient sera possessed specific IgG and IgE antibodies to membranous components. The detergent octyl-beta-D-glucopyranoside was used to extract membrane vesicle components (MC). The enzyme profile of MC compared with cell sap components (CS) showed differences in types of enzymes. Two-dimensional polyacrylamide gel electrophoretic analyses of MC and CS detected components shared as well as unique to each fraction. In crossed immunoelectrophoresis using both rabbit antisera raised to MC and ABPA patient sera, 5 peaks were detected, while analysis of CS using rabbit antisera raised to CS produced 20 major peaks. Immunoelectrophoresis and double immunodiffusion data supported the crossed immunoelectrophoretic data: MC differed from CS. Enzyme-linked immunosorbent assay indicated high specific IgG and IgE antibody levels to MC in ABPA patient sera. Crossed immuno-affinoelectrophoresis with concanavalin A partially characterized the MC, which consist of components which have glycoprotein elements (i.e., containing alpha-D-glucose or alpha-D-mannose).

Electrophoretic and serological analyses of cytoplasmic antigens from Aspergillus fumigatus during growth of conidia to mature mycelia.

The changes of cytoplasmic components concomitant with conidium to mature mycelium growth of Aspergillus fumigatus strain Ag 507 were analysed by one- and two-dimensional polyacrylamide gel electrophoresis (SDS-PAGE; 2-DE). SDS-PAGE monitored molecular weight differences between components of cytosol preparations obtained from conidia and those through 96 h of mycelial growth. 2-DE analyses indicated that some components characteristic of mature cytosol begin to appear by 7 h. Cytoplasmic preparations absorbed with rabbit immunoglobulins raised to mature cytosol were analysed by 2-DE. Conidia cytosol components were not absorbed to a great degree, unlike those from later stages of mycelial growth, which indicates that cytosol components may be changed and/or synthesized de novo during growth of the fungus. Analysis of the cytosol preparations by fused rocket immunoelectrophoresis showed that some components are synthesized in different amounts at various times during growth: 3, 4, 7, 8, and 18 h of growth, components begin to appear that may be synthesized de novo. Enzyme-linked immunosorbent assay with rabbit antiserum to mature cytosol and cytosol preparations obtained from conidia through 96 h of growth, indicated differences of molecular structures between the cytosol preparations. The anticytosol IgG and IgE titers of sera from patients with allergic bronchopulmonary aspergillosis were both elevated and fluctuated with each preparation. The specific IgG and IgE titers both appeared to be elevated with cytosol preparations obtained from 4, 5, 7, and 9 h of growth and highest against the 96 h preparation.

Antigens of Aspergillus fumigatus. III. Comparative immunochemical analyses of clinically relevant aspergilli and related fungal taxa.

Cell sap (CS) of the pathogenic fungus Aspergillus fumigatus strain Ag-507 was fractionated by Sephadex G-200 column chromatography. A protein fraction designated CS3 was partially characterized by two dimensional electrophoresis (2-DE) and analytical ultracentrifugation. CS3 consisted mainly of low molecular weight components (14 K-43 K) of the whole CS, and produced one peak in analytical ultracentrifugation with an Sapp of 4.25. CS3 was demonstrated to be different from a previously characterized CS fraction designated as CS2, by 2-DE, and by CS2 and CS3 specific antisera. CS3 gave precipitin reactions with three aspergilloma patient sera and 100% of sera from allergic bronchopulmonary aspergillosis (ABPA) patients. Significantly, three ABPA patient sera reacted with CS3 and not CS2. The CS of A. fumigatus strains Ag-515 and Ag-534, were also examined for the presence of CS3 components as were CS preparations of five additional Aspergillus species; A. flavus, A. fischeri, A. terreus, Neosartorya (Aspergillus) fennelliae, and A. niger, and three fungal taxa; Penicillium notatum, Candida albicans, and Saccharomyces cerevisiae. 2-DE, immunoelectrophoresis and double diffusion (DD) analyses of the CS preparations provided complementary information. The immunochemical similarity of CS2 and CS3 components of different aspergilli appears to reflect the taxonomic relatedness of the aspergilli. Additionally, aspergilli exhibiting CS2 and CS3 components most similar to A. fumigatus strain Ag-507 are more frequently isolated from aspergillosis patients. There may be an association of these components with incidence of involvement of the organisms in aspergillosis. DD analysis of the cross-reactivity of CS of all taxa with ABPA and aspergilloma patient sera supported the 2-DE and absorption data.

Ultrastructural localization of cellulase in Trichoderma reesei using immunocytochemistry and enzyme cytochemistry.

Two components of the cellulase complex (E.C. 3.2.1.4) of the fungus Trichoderma reesei were localized at the ultrastructural level. Immunocytochemistry and enzyme cytochemistry demonstrated that cellobiohydrolase and beta-1,4 glucanase were localized within cisternae of endoplasmic reticulum and within membrane complexes of cellulose-grown hyphae. Both enzymes were also present in the culture medium. Glucose-grown control hyphae lacked enzyme-specific staining, and no enzyme activity was detected in the growth medium.

Antigens of Aspergillus fumigatus. II. Electrophoretic and clinical studies.

Cell sap (CS) and culture filtrate (CF) preparations of Aspergillus fumigatus strains Ag-507, Ag-515, and Ag-534 were analysed by two dimensional electrophoresis (2-DE; i.e., first dimension isoelectric focusing, second dimension sodium dodecyl sulphate gradient pore gel), which enabled detection of strain- and species-specific components. In CS preparations it was shown that CS2, a fraction isolated from strain Ag-507 by gel filtration, consists of the major protein components in the CS of the three A. fumigatus strains tested. Culture filtrate preparations of the three A. fumigatus strains analysed by 2-DE exhibited patterns dissimilar to the CS patterns, as well as to each other, presumably due to proteolysis. Culture filtrate preparations are therefore a less reliable source of standardized antigens than CS preparations. CS2 has a major component with a mol. wt. of approximately 150,000 and an sapp of 6.3 S. CS2 reacts on immunoelectrophoresis, producing one major precipitin arc with aspergilloma or allergic bronchopulmonary aspergillosis (ABPA) patient sera. Antibody titres of the IgG and IgA classes to CS2, as measured by enzyme-linked immunosorbent assay (ELISA), were demonstrated to be similar in aspergilloma and ABPA patients; IgG titres were higher than IgA. Similar titres were also obtained utilizing sera of patients that did or did not exhibit precipitating antibodies to CS2. In the diagnosis of ABPA, skin tests with CS2 were comparable in specificity to currently available commercial preparations. Importantly, CS2 is a standardized major antigenic preparation of the CS of three A. fumigatus strains which has been shown to be diagnostically useful.

Immunochemical characterization of Nocardia asteroides antigens: support for a single species concept.

Nocardia asteroides strains are highly heterogeneous. They show morphological, physiological, and immunological differences. In a previous study, we delineated seven immunotypes of N. asteroides. In the present study, we compared the culture filtrate antigens of these immunotypes by antigen-antibody crossed-immunoelectrophoresis and by rocket electrophoresis. We have also compared the antigen preparations by two-dimensional electrophoresis. While unique components constitute the major portion of the components, the results indicate that similar components are present in the culture filtrates of all strains. This finding supports the view of retaining all the immunotypes in the species Nocardia asteroides rather than designating different species such as N. farcinica and N. sebivorans.