Chitin biosynthesis in protoplasts and subcellular fractions of Aspergillus fumigatus.

The biosynthesis of chitin has been obtained in broken mycelia and protoplasts of the fungus Aspergillus fumigatus. The specific activity of chitin synthase (EC 2.4.1.16) in a membrane preparation from protoplasts derived from the hyphal tips of A. fumigatus was 26.8-fold greater than that of the chitin synthase in broken mycelia, indicating that the active chitin synthase is located primarily in a membrane-bound site at the hyphal tip. Polyoxin D was a potent competitive inhibitor of the enzyme, having Ki 5.2 +/- 0.8 micron with respect to the natural substrate UDP-N-acetyl-D-glucosamine, which has Km 1.58 mM.     

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.     

The preparation and partial characterization of antigenic fractions obtained from the mycelial walls of several Aspergillus species

Extracts with immunological activity were prepared from Aspergillus fumigatus, A. flavus, A. terreus, A. niger and A. nidulans. In each case crude mycelial wall was extracted with an aqueous solution of Triton X-100 giving detergent-soluble material. Further fractionation was achieved by removing the detergent from this solution; the resultant precipitate was removed by centrifugation, and the aqueous supernatant was used as a source of soluble antigens. The sensitivity of these preparations was compared with that of water-soluble antigenic material, prepared from whole macerated mycelium, by double diffusion and counterimmunoelectrophoresis using homologous antisera and sera from patients suffering from aspergilloma and allergic bronchopulmonary aspergillosis. The selectivity of these antigenic preparations was monitored with heterologous antisera raised in rabbits. Batch variability was analysed for one strain of A. fumigatus using chemical and immunological methods. The nature of the antigenic sites involved in these reactions was investigated by studying the susceptibility of the preparations to proteolytic hydrolysis, periodate oxidation and concanavalin A treatment. The total protein and carbohydrate content of each fraction was determined and the constituent sugars analysed in an attempt to correlate chemical composition with antigenic activity.     

The effect of beta-glucuronidase and chitinase on the cell wall of Aspergillus niger and Aspergillus fumigatus.

The effects of beta-glucuronidase and chitinase have been tested on the hydrolysis of the cell walls of the economically important fungi, Aspergillus niger and Aspergillus fumigatus. The extent of wall hydrolysis was measured by assaying for total reducing sugars, N-acetyl sugars and protoplast production. Maximum reducing sugar release was attained after 40 min incubation, both with beta-glucuronidase supplemented with chitinase and beta-glucuronidase alone, whereas N-acetyl sugar release reached a maximum at 80 min incubation. beta-Glucuronidase was effective in releasing protoplasts from both species of Aspergillus. This release was enhanced by adding chitinase to the incubation medium at 0 and 20 min, but with addition at 60, 80 and 100 min increase in protoplast yield was much reduced. The results of re-incubation experiments with chitinase suggest that this enzyme may in some way be inhibited during the later stages of incubation. Pronase used in combination with beta-glucuronidase slightly enhanced protoplast release.     

Differences among the cell wall galactomannans from Aspergillus wentii and Chaetosartorya chrysella and that of Aspergillus fumigatus

The alkali extractable and water-soluble cell wall polysaccharides F1SS from Aspergillus wentii and Chaetosartorya chrysella have been studied by methylation analysis, 1D- and 2D-NMR, and MALDI-TOF analysis. Their structures are almost identical, corresponding to the following repeating unit: [--> 3)-beta-D-Gal f -(1 --> 5)-beta-D-Gal f-(1 -->]n --> mannan core. The structure of this galactofuranose side chain differs from that found in the pathogenic fungus Aspergillus fumigatus, in other Aspergillii and members of Trichocomaceae: [--> 5)-beta-D-Gal f-(1 -->]n --> mannan core. The mannan cores have also been investigated, and are constituted by a (1 --> 6)-alpha-mannan backbone, substituted at positions 2 by chains from 1 to 7 residues of (1 --> 2) linked alpha-mannopyranoses.     

Differences among the cell wall galactomannans from Aspergillus wentii and Chaetosartorya chrysella and that of Aspergillus fumigatus

The alkali extractable and water-soluble cell wall polysaccharides F1SS from Aspergillus wentii and Chaetosartorya chrysella have been studied by methylation analysis, 1D- and 2D-NMR, and MALDI-TOF analysis. Their structures are almost identical, corresponding to the following repeating unit: [→ 3)-β-D-Galf-(1 → 5)-β-D-Galf-(1 →] _n → mannan core. The structure of this galactofuranose side chain differs from that found in the pathogenic fungus Aspergillus fumigatus, in other Aspergillii and members of Trichocomaceae: [→ 5)-β-D-Galf-(1 →] _n → mannan core. The mannan cores have also been investigated, and are constituted by a (1 → 6)-α-mannan backbone, substituted at positions 2 by chains from 1 to 7 residues of (1 → 2) linked α-mannopyranoses. Published in 2004. This revised version was published online in July 2006 with corrections to the Cover Date.     

Restriction analysis of an amplified rodA gene fragment to distinguish Aspergillus fumigatus var. ellipticus from Aspergillus fumigatus var. fumigatus

A previous multidisciplinary study indicated that gliotoxin-producing Aspergillus fumigatus Fresen. isolates from silage commodities mostly belonged to its variant A. fumigatus var. ellipticus Raper & Fennell. Sequence analysis revealed the presence of a single nucleotide polymorphism at five positions in a fragment of the rodA gene (coding for a hydrophobin rodletA protein) between Aspergillus fumigatus var. fumigatus and Aspergillus fumigatus var. ellipticus. A method was developed to distinguish these two types of isolates based on restriction analysis of this rodA gene fragment using the HinfI restriction enzyme. In addition, in silico analysis of 113 rodA gene fragments retrieved from GenBank was performed and confirmed the suitability of this method. In conclusion, the method developed in this study allows easy distinction between A. fumigatus var. fumigatus and its variant ellipticus. In combination with the earlier developed PCR-restriction fragment length polymorphism method of Staab et al. (2009, J Clin Microbiol 47: 2079), this method is part of a sequencing-independent identification scheme that allows for rapid distinction between similar species/variants within Aspergillus section Fumigati, specifically A. fumigatus, A. fumigatus var. ellipticus, Aspergillus lentulus Balajee & K.A. Marr, Neosartorya pseudofischeri S.W. Peterson and Neosartorya udagawae Y. Horie, Miyaji & Nishim.     

Sterol composition of itraconazole-resistant and itraconazole-susceptible isolates of Aspergillus fumigatus

Sterol composition of four clinical isolates of Aspergillus fumigatus resistant to itraconazole was determined by gas chromatography--mass spectrometry and compared with that of four susceptible strains. For all strains, the major sterol was ergosterol. Sterol compositions were qualitatively and quantitatively similar for the resistant and susceptible strains. These results suggest that itraconazole resistance is not related, for the strains studied, to alterations in the ergosterol synthesis pathway.     

Aspergillus fumigatus RasA regulates asexual development and cell wall integrity

The Ras family of proteins is a large group of monomeric GTPases. Members of the fungal Ras family act as molecular switches that transduce signals from the outside of the cell to signaling cascades inside the cell. A. fumigatus RasA is 94% identical to the essential RasA gene of Aspergillus nidulans and is the Ras family member sharing the highest identity to Ras homologs studied in many other fungi. In this study, we report that rasA is not essential in A. fumigatus, but its absence is associated with slowed germination and a severe defect in radial growth. The DeltarasA hyphae were more than two times the diameter of wild-type hyphae, and they displayed repeated changes in the axis of polarity during hyphal growth. The deformed hyphae accumulated numerous nuclei within each hyphal compartment. The DeltarasA mutant conidiated poorly, but this phenotype could be ameliorated by growth on osmotically stabilized media. The DeltarasA mutant also showed increased susceptibility to cell wall stressors, stained more intensely with calcofluor white, and was refractory to lysing enzymes used to make protoplasts, suggesting an alteration of the cell wall. All phenotypes associated with deletion of rasA could be corrected by reinsertion of the wild-type gene. These data demonstrate a crucial role for RasA in both hyphal growth and asexual development in A. fumigatus and provide evidence that RasA function is linked to cell wall integrity.     

The Volatome of Aspergillus fumigatus

Early detection of invasive aspergillosis is absolutely required for efficient therapy of this fungal infection. The identification of fungal volatiles in patient breath can be an alternative for the detection of Aspergillus fumigatus that still remains problematic. In this work, we investigated the production of volatile organic compounds (VOCs) by A. fumigatusin vitro, and we show that volatile production depends on the nutritional environment. A. fumigatus produces a multiplicity of VOCs, predominantly terpenes and related compounds. The production of sesquiterpenoid compounds was found to be strongly induced by increased iron concentrations and certain drugs, i.e., pravastatin. Terpenes that were always detectable in large amounts were α-pinene, camphene, and limonene, as well as sesquiterpenes, identified as α-bergamotene and β-trans-bergamotene. Other substance classes that were found to be present in the volatome, such as 1-octen-3-ol, 3-octanone, and pyrazines, were found only under specific growth conditions. Drugs that interfere with the terpene biosynthesis pathway influenced the composition of the fungal volatome, and most notably, a block of sesquiterpene biosynthesis by the bisphosphonate alendronate fundamentally changed the VOC composition. Using deletion mutants, we also show that a terpene cyclase and a putative kaurene synthase are essential for the synthesis of volatile terpenes by A. fumigatus. The present analysis of in vitro volatile production by A. fumigatus suggests that VOCs may be used in the diagnosis of infections caused by this fungus.     

The mitogen-activated protein kinase MpkA of Aspergillus fumigatus regulates cell wall signaling and oxidative stress response.

Mitogen-activated protein kinase (MAPK) signaling pathways are involved in the regulation of various cellular responses in eukaryotes. In fungal pathogens they are of special interest because of their possible contribution to pathogenicity. Bioinformatic analysis of the genome of the most prevalent airborne human pathogenic fungus Aspergillus fumigatus, revealed the presence of four distinct MAPK-encoding genes. Here, we present the detailed functional analysis of one of these MAPKs, MpkA. Comparative analysis revealed similarities of MpkA with MAPKs involved in cell wall integrity signaling of other fungi. Accordingly, the analysis of mpkA deletion mutants revealed severe sensitivity of the mutants against cell wall active compounds, drastical alterations of the fungal morphology and increased resistance against oxidative stress. The expression of mpkA was induced by cell wall damaging conditions. Despite its involvement in cell wall signaling no influence on virulence of the deletion of mpkA was observed in a murine infection model.     

Electrofusion of protoplasts from Aspergillus nidulans

Abstract Electrical parameters were determined and quantified for the stimulation of the optimum alignment and fusion of Aspergillus nidulans protoplasts. In a non-homogeneous alternating electrical field A. nidulans protoplasts aligned to form pearl chains associated with the electrodes of the fusion chamber. Most protoplasts were in pearl chains in an alignment field frequency of 3.0 MHz but maximum pair formation occurred at 1.0 MHz. At a field strength between 100 and 1000 V · cm⁻¹ pearl chain formation occurred with minimal protoplast rotation or lysis. The application of DC pulses resulted in protoplast fusion. Most fusion events were observed after two 500 V · cm⁻¹ DC pulses with a 0.5 s interpulse period. Using 1 × 10³ protoplasts · cm⁻³ in a 7 μm fusion chamber a maximum of 17.2 ± 2.0% fusion events were achieved.     

The chemical composition of the membranes of protoplasts and L-forms of Staphylococcus

Membrane fractions were prepared from Staphylococcus aureus H and 100 after dissolution of the cell walls by a lytic enzyme from Streptomyces griseus. Membranes were also prepared from the L-forms derived from the same strains. The membranes were analysed for protein, lipid, carbohydrate and RNA contents, and the fatty acid composition of the lipids was determined. A branched-chain saturated C(15) acid was the major component in all samples, and the correspondence between L-forms and parent bacteria was fairly close. The lipids were separated into non-polar-lipid, glycolipid and phospholipid fractions; the L-forms contained a little more neutral lipid and much more glycolipid than the parent bacteria. In all membranes the glycolipid, which accounted for all the carbohydrate present, was a diglucosyl diglyceride. The major phospholipids of the protoplast membranes were phosphatidylglycerol and some lipoamino acids (lysine and a little alanine). On the other hand, diphosphatidylglycerol was the chief phospholipid found in L-form membranes.     

Molecular organization of the alkali-insoluble fraction of Aspergillus fumigatus cell wall

Physical and biological properties of the fungal cell wall are determined by the composition and arrangement of the structural polysaccharides. Cell wall polymers of fungi are classically divided into two groups depending on their solubility in hot alkali. We have analyzed the alkali-insoluble fraction of the Aspergillus fumigatus cell wall, which is the fraction believed to be responsible for fungal cell wall rigidity. Using enzymatic digestions with recombinant endo-beta-1,3-glucanase and chitinase, fractionation by gel filtration, affinity chromatography with immobilized lectins, and high performance liquid chromatography, several fractions that contained specific interpolysaccharide covalent linkages were isolated. Unique features of the A. fumigatus cell wall are (i) the absence of beta-1,6-glucan and (ii) the presence of a linear beta-1, 3/1,4-glucan, never previously described in fungi. Galactomannan, chitin, and beta-1,3-glucan were also found in the alkali-insoluble fraction. The beta-1,3-glucan is a branched polymer with 4% of beta-1,6 branch points. Chitin, galactomannan, and the linear beta-1, 3/1,4-glucan were covalently linked to the nonreducing end of beta-1, 3-glucan side chains. As in Saccharomyces cerevisiae, chitin was linked via a beta-1,4 linkage to beta-1,3-glucan. The data obtained suggested that the branching of beta-1,3-glucan is an early event in the construction of the cell wall, resulting in an increase of potential acceptor sites for chitin, galactomannan, and the linear beta-1,3/1,4-glucan.     

Fumitoxins, new mycotoxins from Aspergillus fumigatus Fres.

Extracts of cultures of Aspergillus fumigatus isolated from silage were lethal to chicken embryos. Using this test and thin-layer chromatography, four UV-absorbing toxins, designated as fumitoxins A, B, C and D, were isolated. Analysis and mass spectrometry of crystallized fumitoxin A, the most abundant in the extract, established its molecular formula to be C31H42O8. Infrared, UV spectroscopy, and chemical reactions suggested that fumitoxin A is a steroid. Fumitoxins appear to be clearly different from the previously described toxins recognized in A. fumigatus.