Analysis of Aspergillus fumigatus catalases possessing antigenic activity.

Analysis of Aspergillus fumigatus water soluble fractions by electrophoresis on non-denaturing polyacrylamide gels (PAGE) showed the presence of at least three catalase bands. They were designated F, S1 and S2 in order of descending electrophoretic mobility with respect to the anode. The multiple enzyme forms appear to be distinct in their physicochemical properties. Enzyme bands S1 and S2 were simple catalases; the F band had an additional peroxidase function. All of the components were antigenic and differed in their binding to specific antibodies raised in rabbits with separate fractions of A. fumigatus mycelium. When serum from patients with aspergilloma, allergic bronchopulmonary aspergillosis, cystic fibrosis and chronic asthma were pre-incubated with A. fumigatus antigens and analysed by PAGE, 17 of 26 samples either abolished or reduced catalase activity. Enzyme F was a non-Concanavalin A (ConA)-binding antigen; the S1 and S2 enzymes were ConA-binding glycoprotein antigens. The major catalase band present in A. niger preparations represented only a minor component in A. fumigatus.     

Partial characterization of a rapidly released antigenic/allergenic component (Ag 5) of Aspergillus fumigatus

An antigenic component of Aspergillus fumigatus, which was responsible for inducing an initial early antibody response in rabbits immunized with A. fumigatus germlings, has been identified and partially characterized. By immunofluorescent antibody and ELISA techniques, this antigen was demonstrated to be present on the germling surface, although it was also detected in supernatants of conidia/germlings within 1 hour and was present in all shake and stationary culture-filtrate extracts. By incorporation of a monospecific antiserum to this component in an intermediate gel of the reference self-crossed radioimmunoelectrophoresis pattern for allergic bronchopulmonary aspergillosis sera, it was recognized as Ag 5 and was also demonstrated to bind specific IgE. Further immunochemical analyses have revealed that Ag 5 is relatively heat labile, does not bind to concanavalin A, and has a molecular weight of approximately 35 kd. This rapidly released antigenic/allergenic component may play an important role in the initiation of immunologic responses in patients with allergic bronchopulmonary aspergillosis.     

Cloning and disruption of the antigenic catalase gene of Aspergillus fumigatus.

Aspergillus fumigatus possesses two catalases (described as fast and slow on the basis of their electrophoretic mobility). The slow catalase has been recognized as a diagnostic antigen for aspergillosis in immunocompetent patients. The antigenic catalase has been purified. The enzyme is a tetrameric protein composed of 90-kDa subunits. The corresponding cat1 gene was cloned, and sequencing data show that the cat1 gene codes for a 728-amino-acid polypeptide. A recombinant protein expressed in Pichia pastoris is enzymatically active and has biochemical and antigenic properties that are similar to those of the wild-type catalase. Molecular experiments reveal that CAT1 contains a signal peptide and a propeptide of 15 and 12 amino acid residues, respectively. cat1-disrupted mutants that were unable to produce the slow catalase were as sensitive to H2O2 and polymorphonuclear cells as the wild-type strain. In addition, there was no difference in pathogenicity between the cat1 mutant and its parental cat1+ strain in a murine model of aspergillosis.     

Characterization of the major Woronin body protein HexA of the human pathogenic mold Aspergillus fumigatus

In filamentous fungi, the septal pore controls the exchange between neighbouring hyphal compartments. Woronin bodies are fungal-specific organelles that plug the pore in case of physical damage. The Hex protein is their major and essential component. Hex proteins of different size are predicted in the data base for pathogenic and non-pathogenic Aspergillus species. However, using specific monoclonal antibodies, we identified 2 dominant HexA protein species of 20 and 25 kDa in A. fumigatus, A. terreus, A. nidulans, and A. oryzae. HexA and Woronin bodies were found in A. fumigatus hyphae, but also in resting conidia. Using monoclonal antibodies, a GFP-HexA fusion protein, and an RFP protein fused to the putative peroxisomal targeting sequence of HexA, we analyzed the spatial localization and dynamics of Woronin bodies in A. fumigatus as well as their formation from peroxisomes. In intact hyphae, some Woronin bodies were found in close proximity to the septal pore, while the majority was distributed in the cytoplasm. Septum-associated Woronin bodies show a minimal lateral movement, while the cytosolic Woronin bodies are highly dynamic. The distribution of Woronin bodies and their co-localization pattern with peroxisomes revealed no evidence that Woronin bodies arise predominantly at the apical tip of A. fumigatus hyphae. We found that Woronin bodies are able to plug septal pores of A. fumigatus in case of damage. Woronin bodies therefore contribute to the stress resistance and potentially also to the virulence of A. fumigatus, which renders them a potential target for future anti-fungal strategies.     

Secretome analysis of Aspergillus fumigatus reveals Asp-hemolysin as a major secreted protein

Surface-associated and secreted proteins represent primarily exposed components of Aspergillus fumigatus during host infection. Several secreted proteins are known to be involved in defense mechanisms or immune evasion, thus, probably contributing to pathogenicity. Furthermore, several secreted antigens were identified as possible biomarkers for the verification of diseases caused by Aspergillus species. Nevertheless, there is only limited knowledge about the composition of the secretome and about molecular functions of particular proteins. To identify secreted proteins potentially essential for virulence, the core secretome of A. fumigatus grown in minimal medium was determined. Two-dimensional gel electrophoretic separation and subsequent MALDI–TOF-MS/MS analyses resulted in the identification of 64 different proteins. Additionally, secretome analyses of A. fumigatus utilizing elastin, collagen or keratin as main carbon and nitrogen source were performed. Thereby, the alkaline serine protease Alp1 was identified as the most abundant protein and hence presumably represents an important protease during host infection. Interestingly, the Asp-hemolysin (Asp-HS), which belongs to the protein family of aegerolysins and which was often suggested to be involved in fungal virulence, was present in the secretome under all growth conditions tested. In addition, a second, non-secreted protein with an aegerolysin domain annotated as Asp-hemolysin-like (HS-like) protein can be found to be encoded in the genome of A. fumigatus. Generation and analysis of Asp-HS and HS-like deletion strains revealed no differences in phenotype compared to the corresponding wild-type strain. Furthermore, hemolysis and cytotoxicity was not altered in both single-deletion and double-deletion mutants lacking both aegerolysin genes. All mutant strains showed no attenuation in virulence in a mouse infection model for invasive pulmonary aspergillosis. Overall, this study provides a comprehensive analysis of secreted proteins of A. fumigatus and a detailed characterization of hemolysin mutants.     

Interactions between conidia of Aspergillus fumigatus and human complement component C3.

Activation and deposition of C3 on Aspergillus fumigatus conidia have been previously demonstrated. This study investigated in further detail the interactions between complement component C3 and the conidia of A. fumigatus. Immunoblotting and 125I-C3 binding studies showed that C3 deposition was rapid (less than 15 min) and parallel to the formation of iC3b. Immunoblotting experiments identified a 54- to 58-kDa conidial protein which binds human complement component C3 and/or a C3 fragment(s). 125I labeling of the outer layer of the conidia demonstrated that this protein doublet was present on the surface of the spore. The further degradation of C3 to low-molecular-mass fragments (40, 37, and 30 kDa), in the absence of plasma, by intact living conidia and a preparation of the outer conidial wall layer indicated the ability of fungal components to cleave C3. These data suggest that interactions between conidia and C3 are not limited only to deposition via activation of the alternative complement pathway; they also include degradation of bound C3.     

Characterization of a second major antigen Ag 13 (antigen C) of Aspergillus fumigatus and investigation of its immunological reactivity.

A second major antigen of Aspergillus fumigatus (Ag 13) has been identified and characterized as a 70 kD, heat-labile component which is able to bind to the lectin, concanavalin A. Ag 13 proved to be identical to the previously recognized C antigen, known to possess chymotryptic activity. A monospecific antiserum to Ag 13 has been affinity purified and used to develop a sandwich ELISA for measuring levels of Ag 13 specific IgG antibodies in patients' sera. Although all ABPA sera (25/25) had significantly raised levels, some aspergilloma sera (2/5) had only very low levels; of the control sera, 10/10 fungal atopics were negative, but 5/12 farmer's lung sera had low but positive values of Ag 13 specific IgG, i.e. sensitivity 100%, specificity 80%. Ag 13 is therefore a diagnostically important component of A. fumigatus and the antigen-specific ELISA may be of importance for the improved detection of disease-specific antibodies.     

AFMP2 encodes a novel immunogenic protein of the antigenic mannoprotein superfamily in Aspergillus fumigatus

We cloned the Aspergillus fumigatus mannoprotein 2 (AFMP2) gene, which encodes a novel immunogenic protein (Afmp2p) of the antigenic mannoprotein superfamily, in A. fumigatus. Sequence analysis revealed that Afmp2p has 510 amino acid residues, with a predicted molecular mass of 51.5 kDa. Afmp2p has a putative N-terminal signal peptide, a putative C-terminal glycosylphosphatidylinositol membrane attachment signal sequence, and an upstream GAA cleavage site commonly used for cytoplasmic membrane attachment and implicated in fungal cell wall assembly. Upstream of the GAA cleavage site, Afmp2p contains a 302-amino-acid serine- and threonine-rich region as a site for potential O-glycosylation. Within this serine- and threonine-rich region, 13 repeats of ETSTPCE(T)(n) were observed. Western blot analysis of Afmp2p in A. fumigatus fungal cell lysate and culture supernatant and immunogold staining and electron microscopy showed that Afmp2p is predominantly secreted into the culture supernatant, whereas only minimal amounts can be detected in the cell lysate and cell wall. Finally, it was observed that patients with aspergilloma and invasive aspergillosis due to A. fumigatus develop a specific antibody response against recombinant Afmp2p. The abundance of Afmp2p in secreted form, its minimal cross-reactivity with Afmp1p, and the presence of an antibody response against Afmp2p in patients with A. fumigatus infections suggest that Afmp2p is a good candidate for complementing Afmp1p in serodiagnosis of A. fumigatus infections.     

rodletless mutants of Aspergillus fumigatus.

Conidia of Aspergillus fumigatus adhere in vitro to host proteins and cells via the outer cell wall layer. The rodA gene of A. fumigatus was cloned by homology with the rodA gene of Aspergillus nidulans, which is involved in the structure of the rodlets characteristic of the surface layer. The A. fumigatus RODA protein sequence has 85% similarity to that of A. nidulans RODA; the sequence codes for a hydrophobin, a low-molecular-weight protein moderately hydrophobic and rich in cysteines. The gene was disrupted with the hygromycin B resistance gene. By transformation of protoplasts with the disrupted gene, RodA- mutants were generated. These mutants are deficient in the ability to disperse their conidia; their conidia lack the rodlet layer and are hydrophilic. The adhesion of the rodletless conidia to collagen and bovine serum albumin was lower than that of the wild type; in contrast, there was no difference between RodA- and RodA+ conidia in adhesion to pneumocytes, fibrinogen, and laminin, suggesting that RODA is not the receptor for these cells and proteins. RodA- conidia were pathogenic for mice.     

PCR identification system for the genus Aspergillus and three major pathogenic species: Aspergillus fumigatus, Aspergillus flavus and Aspergillus niger.

A PCR system was developed that allowed recognition of three major pathogenic Aspergillus species, namely A. fumigatus, A. niger and A. flavus, in isolates obtained from clinical specimens. The primer pair for PCR was designed from conserved sequences of internal transcribed spacer 1 (ITS1) ribosomal DNA and its flanking regions. Products 521 bp in size were successfully amplified by PCR from the seven Aspergillus species most frequently encountered as opportunistic pathogens, and the three most commonly significant species were identified by separate PCR reactions or nested PCR based on use of species-specific primers. To our knowledge, this is first report of identification of the second and third most frequent pathogenic Aspergillus species using specific PCR amplification. The PCR based identification system reported here will be a powerful tool to control difficult pulmonary fungal infections and to speed the application of effective treatment.     

Characterization of immunologically important antigens and allergens of Aspergillus fumigatus

Using a variety of immunochemical methods, including quantitative immuno-electrophoretic techniques, combined with gel filtration and iso-electric focusing, and production of monospecific antisera for identification and affinity purification, 4 major components of Aspergillus fumigatus have now been partially characterized. Numbering of these was derived from a reference allergic bronchopulmonary aspergillosis (ABPA) self-crossed radio-immuno-electrophoresis pattern of reactivity. Two major intracellular/cytoplasmic, concanavalin A (Con A)-binding antigens, Ag 7 and Ag 13, of molecular weights 150-200 and 70 kilodaltons (kD), respectively, were confirmed to be of importance for both ABPA and aspergilloma in specific sandwich enzyme-linked immunosorbent assays. A rapidly released component, Ag 5, of molecular weight 35 kD, proved both antigenic and allergenic, with aspergilloma patients having especially high-titre IgG antibodies. The major allergenic component Ag 3, of molecular weight 24 kD by gel filtration and 18 kD by SDS-PAGE was, like Ag 5, relatively heat-labile and non-Con-A-binding. Interestingly, T cell clones have been identified which respond primarily to an 18-kD fraction.     

Characterization of a monoclonal antibody against concanavalin A binding antigen of Aspergillus fumigatus

A monoclonal antibody was produced against a Concanavalin A (Con A) binding major epitope of Aspergillus fumigatus using a novel method of immunization. The antigen was purified using monoclonal antibody affinity chromatography reacted with specific antibodies present in human sera. Both allergic bronchopulmonary aspergillosis and aspergilloma showed high levels of antibody, against this purified antigen, when compared to normal controls. Similar results were obtained when the monoclonal antibody was used in a capture antigen assay. The antibody reacted with several A. fumigatus extracts in rocket electrophoresis demonstrating a single precipitin arc, which disappeared when Con A intermediate gel was used. This monoclonal antibody demonstrated reactivity only with cytoplasmic components of hyphae and spores of A. fumigatus, when a colloidal gold was used as a probe in immunoelectron microscopy.     

Acquired immunity to Aspergillus fumigatus.

In mice preinfected with Aspergillus fumigatus for greater than or equal to days, administration of cortisone failed to stimulate mycelial growth. The liver and heart of mice preinfected for 14 days resisted infection after a cortisone + conidia challenge.     

Recombinant expression and antigenic properties of a 32-kilodalton extracellular alkaline protease, representing a possible virulence factor from Aspergillus fumigatus.

A 32-kDa nonglycosylated alkaline protease (EC 3.4.1.14) with elastolytic activity, secreted by the opportunistic pathogen Aspergillus fumigatus ATCC 42202, is suggested to be a virulence factor of this fungus. The enzyme is a serine protease of the subtilisin family, and its cDNA nucleotide sequence has recently been reported. We have cloned the cDNA encoding the mature protease into a high-level Escherichia coli expression plasmid and produced the recombinant protease as a fusion protein with a six-adjacent-histidine affinity tag at the carboxy terminus. Subsequently, the recombinant protease was purified to homogeneity, with affinity chromatography yielding 30 to 40 mg of recombinant protease per liter of E. coli culture. Refolded recombinant protease, in comparison with native protease, demonstrated weak enzymatic activity but similar immunochemical characteristics as analyzed by antigen-specific enzyme-linked immunosorbent assay (ELISA), competition ELISA, and immunoblotting assays. To assess the allergenic potential of the protease, sera from patients with allergic bronchopulmonary aspergillosis and sera from healthy control individuals were analyzed by ELISA and immunoblotting techniques. Sera from patients with allergic bronchopulmonary aspergillosis did not have protease-specific immunoglobulin E (IgE) antibodies and, remarkably, did not show significantly elevated protease-specific IgG antibody levels compared with those in sera from healthy control individuals. This suggests that the alkaline protease from A. fumigatus does not elicit IgE antibodies and has weak immunogenicity, a property which may explain fungus persistence in allergic individuals.     

Immunochemical characterization of Aspergillus fumigatus antigens.

Culture filtrate antigens of Aspergillus fumigatus Ag 534 were purified by preparative isoelectric focusing and affinity chromatography. One of the pooled antigen fractions from the preparative isoelectric focusing step (pool 2) was passed through a concanavalin A column and yielded two components, designated antigens IIa and IIb. Antigen IIb reacted more strongly than antigen IIa with all of the aspergilloma and allergic bronchopulmonary aspergillosis sera tested by enzyme-linked immunosorbent assay. The glycoprotein nature of antigen IIb was shown by the concanavalin A binding properties and staining reactions of the components.     

Fetuin A, a serum component, promotes growth and biofilm formation by Aspergillus fumigatus

Aspergillus fumigatus is an all-important pathogenic fungus and is known for its angiotropism. When it invades human organs, A. fumigatus makes direct contact with blood and its components by causing inflammation and invading vascular structures. To learn the effect of its contact with blood on the development of infection, we examined the effect of serum on A. fumigatus growth. In Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, hyphal tip growth was accelerated, forming a thickened and well-networked biofilm associated with extracellular matrix, and fetuin A was identified as the active component in the serum that accelerates fungal growth leading to formation of a community. These results suggest that fetuin A is a novel accelerator of the growth of A. fumigatus and that it participates in the formation of thick biofilm.     

Catalases of Aspergillus fumigatus

Upon infection of a host, the pathogenic fungus Aspergillus fumigatus is attacked by the reactive oxygen species produced by phagocytic cells. Detoxification of hydrogen peroxide by catalases was proposed as a way to overcome this host response. A. fumigatus produces three active catalases; one is produced by conidia, and two are produced by mycelia. The mycelial catalase Cat1p was studied previously. Here we characterized the two other catalases, their genes, and the phenotypes of gene-disrupted mutants. CatAp, a spore-specific monofunctional catalase, is resistant to heat, metal ions, and detergent. This enzyme is a dimeric protein with 84.5-kDa subunits. The 749-amino-acid polypeptide exhibits high levels of similarity to the Aspergillus nidulans CatA catalase and to bacterial catalase HPII of Escherichia coli. In spite of increased sensitivity to H(2)O(2), killing of DeltacatA conidia by alveolar macrophages and virulence in animals were similar to the killing of conidia by alveolar macrophages and virulence in animals observed for the wild type. In contrast to the Cat1p and CatAp catalases, the mycelial Cat2p enzyme is a bifunctional catalase-peroxidase and is sensitive to heat, metal ions, and detergent. This enzyme, an 82-kDa monomer, is homologous to catalase-peroxidases of several fungi and bacteria. Surprisingly, mycelium of the double Deltacat1Deltacat2 mutant with no catalase activity exhibited only slightly increased sensitivity to H(2)O(2) and was as sensitive to killing by polymorphonuclear neutrophils as mycelium of the wild-type strain. However, this mutant exhibited delayed infection in the rat model of aspergillosis compared to infection by the wild-type strain. These results indicate that conidial catalase is not a virulence factor and that mycelial catalases transiently protect the fungus from the host.     

Conditional virulence of a p-aminobenzoic acid-requiring mutant of Aspergillus fumigatus.

The induced auxotrophy for p-aminobenzoic acid (PABA) resulted in a complete loss of virulence of Aspergillus fumigatus for normal as well as cortisone-treated mice. The PABA-requiring mutant of A. fumigatus survived in vivo for 4 to 7 days without causing any infection. However, it showed conditional virulence in animals receiving PABA in very small quantities. Repeated inoculations of the viable spores of the avirulent mutant strain gave favorable results in building immunity against intravenous challenge of the virulent strain. The immunogenicity of the PABA-requiring mutant was comparable with that of a wild strain of the fungus in agar gel double-diffusion tests using clinical and hyperimmune sera and in skin tests on patients with allergic bronchopulmonary aspergillosis.     

Isolation and characterization of a secreted metalloprotease of Aspergillus fumigatus.

A metalloprotease (MEP) secreted by Aspergillus fumigatus was isolated from an alkaline protease-deficient mutant after the fungus was cultivated in the presence of collagen as the sole nitrogen and carbon source. The enzyme was purified 50-fold from the culture supernatant after adsorption to hydroxylapatite and carboxy-methyl-Sephadex and after gel filtration. The molecular mass was determined to be 40 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric point was estimated at pH 5.5 by isoelectric focusing. Reducing agents and divalent cations strongly inhibited enzyme activity, whereas nonionic detergents had no effect. A. fumigatus MEP was totally inhibited by EDTA, 1,10-phenanthroline, and phosphoramidon but not by inhibitors specific for serine, aspartate, and cysteine proteases. MEP is not able to cleave elastin and is thermosensitive. Sera from patients suffering from aspergilloma reacted with MEP in Western blotting (immunoblotting) analyses, suggesting that MEP promotes an antigenic response in these patients.