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Cell wall α1-3glucans induce the aggregation of germinating conidia of Aspergillus fumigatus
Fungal Genetics and Biology 47 (2010) 707–712
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Cell wall a1-3glucans induce the aggregation of germinating conidia of Aspergillus fumigatus Thierry Fontaine a,*, Anne Beauvais a, Céline Loussert b, Benoît Thevenard a,1, Claus. C. Fulgsang c, Naohito Ohno d, Cécile Clavaud a, Marie-Christine Prevost b, Jean-Paul Latgé a a
Unité des Aspergillus, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris cedex 15, France Plateforme de Microscopie Électronique, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris cedex 15, France Novozymes A/S Bagwaerd DK-2880, Denmark d School of Pharmacy, Tokyo University of Pharmacy and Life science, Tokyo 192-0392, Japan b c
a r t i c l e
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Article history: Received 8 March 2010 Accepted 27 April 2010 Available online 4 May 2010 Keywords: Aspergillus fumigatus Conidial germination a1-3Glucan Cell wall
a b s t r a c t The germination of Aspergillus fumigatus conidia can be divided into four stages: breaking of dormancy, isotropic swelling, establishment of cell polarity, and formation of a germ tube. Swelling of conidia is associated in liquid medium with a multi-cellular aggregation that produced large clumps of conidia. Conidial aggregation can be specifically prevented by the addition of a1-3glucanase. Swollen conidia specifically adhere to insoluble a1-3glucan chains. Electron microscopy studies showed that cell wall a1-3glucan chains became exposed at the cell surface during the swelling. These results demonstrate that cell wall a1-3glucans play an essential role in the aggregation between swollen conidia. Experiments with a1-3glucan coated latex beads show that a1-3glucan chains interacted between them without the requirement of any other cell wall component suggesting that biophysical properties of a1-3glucans are solely responsible for conidial aggregation. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Aspergillus species produce small hydrophobic conidia that are dispersed into the air. Under favourable nutritional conditions, conidia germinate to colonise new environments. Conidial germination can be divided into four steps: breaking of the dormancy, isotropic swelling, establishment of cell polarity and formation of a germ tube (Barhoom and Sharon, 2004; d’Enfert, 1997; Harris and Momany, 2004). In liquid medium, conidial germination of Aspergillus fumigatus conidia was associated with conidial agglutination (Lamarre et al., 2008), that has been described in a number of filamentous fungi many years ago (Metz and Kossen, 1977). Conidial agglutination does not influence the total germination, but increases the size of the pellets produced under shake conditions and affects the use of such filamentous fungi in biotechnology (Kelly et al., 2006; Papagianni, 2004). In Aspergillus niger, a mathe-
Abbreviations: DMSO, dimethylsulfoxide; AS-OxP, periodate oxided alkalisoluble cell wall fraction of A. fumigatus that contains only a1-3glucans; HPAEC, high performance anion exchange chromatography; LGM, lipogalactomannan. * Corresponding author. E-mail address: [email protected] (T. Fontaine). 1 Present address: UR INRA No. 0477, Biochimie bactérienne, Domaine de Vilvert, 78332 Jouy en Josas, France. 1087-1845/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2010.04.006
matic model suggests that conidial aggregation corresponds to an agglomeration step but not a tight intergrowth of particles (Lin et al., 2008), suggesting a cell surface recognition. However, the cell surface molecules involved in this cellular interaction remain unknown. The conidial cell wall is composed of two layers: an external hydrophobic layer containing melanin and rodlet proteins and an internal layer, electron translucent, containing polysaccharides such as a and b-glucans, chitin/chitosan and galactomannan (Maubon et al., 2006). During germination, swelling of conidia requires cell wall remodelling. Indeed, during the isodiametric growth, the conidial surface is dramatically changed. Scanning electron microscopy and by atomic force microscopy have demonstrated that the outer rodlet/melanin layer of the resting conidia is progressively lost and the cell surface layer is changed into a layer of amorphous material unmasking b1-3glucan on the surface of the conidium (Dague et al., 2008; Gersuk et al., 2006; Luther et al., 2007; Rohde et al., 2002). Earlier studies have suggested that glucan exposure or changes in electrical charges and hydrophobicity could be responsible for the agglutination process but no experimental proof was given (Dynesen and Nielsen, 2003; Ryoo and Choi, 1999; Tronchin et al., 1995). In this study we showed that cell wall a1-3glucans, unmasked during germination, are responsible for the aggregation of swollen conidia of A. fumigatus.
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2. Materials and methods
2.4. Isolation of A. fumigatus polysaccharides
2.1. Strains, media, enzymes and chemical compounds
The lipogalactomannan was prepared from mycelium membrane of A. fumigatus (Costachel et al., 2005). The galactosaminogalactan is extracted from the Brian culture medium of A. fumigatus and precipitated with 2.5 vol. of ethanol at 4 °C overnight. After centrifugation (5000g, 10 min), the pellet was washed with 150 mM NaCl. The galactosaminogalactan was extracted by 8 M urea and then dialysed against water and freeze-dried (Delangle, unpublished results). Alkali-insoluble (AI) and alkali-soluble (AS) fractions were prepared from the mycelial cell wall of A. fumigatus as previously described (Fontaine et al., 2000). Cell wall a1-3glucan containing the AS fraction was purified by periodate oxidation. The AS fraction was treated with 100 mM NaIO4 at 4 °C for 5 days in the dark. After the addition of ethylene glycol to destroy the excess of reagent, cell wall fractions were dialysed, reduced with BH4Na, and hydrolysed by Smith degradation (10% acetic acid 100 °C, 1 h). After dialysis against water and freeze-drying, a1-3glucan-oligosaccharides were produced by acetolysis. One gram of periodate-treated AS fraction (AS-OxP) was acetolysed with 100 ml of acetic acid/acetic anhydride/sulphuric acid solution (10/10/1, v/v/v) for 3 days at room temperature under magnetic stirring. The reaction was stopped in a water–ice bath by the addition of pyridine until a neutral pH was achieved. Peracetylated products were extensively dialysed against water at 4 °C and freeze-dried. De-O-acetylation was done by NaOH 0.1 M at room temperature. After centrifugation (5000g for 10 min), the supernatant was neutralised by the addition of acetic acid and fractionated by gel filtration on a G15-Sephadex column (GE Healthcare, 90 130 cm) equilibrated in 0.2% acetic acid and eluted at the flow rate of 7.5 ml/h. Insoluble material was removed by centrifugation, dialysed against water and freeze-dried. The size of acetolysed oligosaccharides was estimated by anion exchange chromatography equipped with a pulsed electrochemical detector (model ICS-3000, Dionex) with a CarboPAC PA-200 column (4.5 250 mm, Dionex) using the following conditions: flow rate 0.35 ml/min; solvent A, NaOH 50 mM; solvent B, NaOAc 500 mM in NaOH 50 mM; gradient initial time to 2 min isocratic elution with 98% A, 2% B; 2–15 min: 2–35% B; 15–35 min 35–100% B; 35–40 min isocratic elution with 100% B (Supplementary Fig. 1).
The fungal strains used in this study, A. fumigatus, Penicillium and Rhizopus species are presented in the Supplementary Table 1. Strains were maintained on 2% malt agar tubes. Mutan (waterinsoluble a-glucan containing mainly a1-3linkages) and mutanase, an a1-3glucanase from Trichoderma harzianum, were obtained as described (Fuglsang et al., 2000). Chitin and laminarin (soluble b1-3glucan) from Laminaria digitata were purchased from Sigma. Curdlan (insoluble b1-3glucan) is a kind gift from Dr. Hidemitsu Kobayashi. Mutanase was purified as previously described (Dekker et al., 2004). Zymolyase T20 from Arthrobacter luteus was purchased from ICN. Jack bean a-mannosidase was purchased from Sigma. Glucanex, a b1-3glucanase cocktail, was purchased from Novo Nordisk Ferment Ltd., (Dittinger, Switzerland). Chitinase A from Serratia macerans, recombinant in Escherichia coli, was a kind gift from Dr. C. Vorgias (Vorgias et al., 1993). Recombinant laminarinase A protein isolated from Thermotoga neapolitana, and expressed in E. coli, a kind gift of Dr. V. Zverlov (Institute of Molecular Genetics, Russian academy of sciences, Moscow), was purified as previously described (Zverlov et al., 1997). Proteinase K was purchased from Roche. Fungal orthologous of AGS genes have been search in the NCBI database (http://www.ncbi.nlm.nih. gov/sutils/genom_table.cgi?organism=fungi). 2.2. Enzyme assays Glycosyl hydrolase activity was quantified in 150 mM sodium acetate pH 5.5. p-Nitrophenyl-a-D-mannoside (Sigma) was used as a substrate for the Jack bean a-mannosidase activity (Bischoff et al., 1990). b1-3Glucanase and a1-3glucanase activities were determined with reduced laminarin or reduced mutan, respectively and quantified with the p-hydroxybenzoic acid hydrazide reagent (Hartland et al., 1996). Chitinase activity was determined using the fluorogenic substrates of methyl-umbeliferiyl-chitobiose (4-MU-(GlcNAc)2, Sigma) at the final concentration of 75 lM. The amount of 4-MU released from the assay reaction was determined at 320 nm excitation and 448 nm emission using a spectrofluorometer. Corresponding activities were summarised in Supplementary Table 2. 2.3. Conidial aggregation assay Conidia were recovered from slants using an aqueous 0.05% Tween 20 solution to obtain a homogeneous suspension. Aggregation assays were done in 24 well plates (TPP, Trasadingen, Switzerland) in 1 ml Sabouraud liquid medium (2% glucose and 1% mycopeptone, Oxoid) containing 0.05% Tween 20 and agitated at 150 rpm at 37 °C for 30 min to 3 h. The presence of 0.05% Tween 20 was required to maintain conidia in suspension in the liquid medium. Agglutination was monitored over time by visual examination. Putative inhibitors of aggregation were added to Sabouraud medium before the inoculation of conidia. Mutan and curdlan are two water-insoluble glucans. To obtain a homogeneous suspension, mutan was first solubilised in DMSO (5 mg/ml) and then precipitated by dilution in water (5 vol.). The precipitate was recovered by centrifugation (5000g, 10 min), washed with water and kept at 4 °C at 5 mg/ml. Curdlan was suspended at 5 mg/ml in water, heated at 65 °C for 30 min and stored at 4 °C. The conidial aggregation assays with Penicillium and Rhizopus strains were done at 25 °C. Conidial aggregate obtained after 3 h of germination were tested for their sensitivity to mutanase, proteinase K, and acidic and basic treatments (100 mM NaOH and 100 mM HCl) that unfold proteins.
2.5. Electron microscopy Resting and 3 h germinating conidia were fixed in 2% paraformaldehyde, 0.2% glutaraldehyde in 0.1 M Sorensen buffer at room temperature for 2 h then overnight at 4 °C. After fixation, free aldehydes were blocked by incubation in 50 mM NH4Cl in PBS for 15 min. Then samples were embedded in 12% gelatin. After solidification, samples were cut into small blocks and then transferred to 2.1 M sucrose in 0.1 M Sorensen buffer for 48 h at 4 °C. Finally, blocks were mounted on pins and frozen in liquid nitrogen. Cryosectioning was done using an UltraCut EM FC6 (Leica, Vienna, Austria) at 110 °C, and 50 nm sections were picked up with 0.8% methylcellulose, 1.26 M sucrose in 0.06 M Sorensen buffer. Immunolabelling of sections on copper grids was done as previously described (Griffith et al., 2008) using a polyclonal antibody directed against a1-3 glucan (Sugawara et al., 2003). The primary antibody was diluted 1:50, then treated with an anti-rabbit F (ab0 )2 goat antibody (diluted 1:25) coupled to 10 nm colloidal gold (BBI) (Beauvais et al., 2007). Sections were observed under 80 kV in a Jeol transmission electron microscope (Jem 1010). 2.6. Coating of glucan to latex beads Twenty milligrams of water-insoluble fraction of acetolysed and borohydride-reduced AS fraction were incubated in 1 ml of
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50 mM IO4Na for 30 min on ice. After addition of 100 ll of glycerol, oxidised glucans were desalted by centrifugation and washed with water. Thirty-four milligrams of biotin-hydrazide (Pierce) were dissolved in 3 ml of DMSO, added to the glucan pellet and incubated for 3 h at room temperature. After the addition of 3 ml of 0.2 M NaOAc pH 5.5, the mixture was left overnight at room temperature, then dialysed against water and freeze-dried. Two milligrams of biotinylated a1-3glucan was dissolved in 500 ll of DMSO, then diluted with 500 ll of water and incubated with 50 ll of streptavidin-activated latex beads (Sigma, 1 lm, 1010 beads/ml, red labelled) for 90 min at room temperature. After addition of 500 ll of water, excess of biotinylated glucans was removed by centrifugation (10 min, 8000g). Latex beads were then washed once with 30% of DMSO and twice with water and kept at 4 °C. b1-3Glucan coated latex beads were used as negative control in the aggregation assay. b1-3Glucan chains were obtained by acetolysis as previously described (Martin-Cuadrado et al., 2008). Biotinylation and coupling to streptavidin-activated latex beads were done as described for a1-3glucans. The coating of glucan on latex beads was confirmed by immunofluorescence using specific antibodies against a1-3glucan (Sigma, mouse IgM MOPCk 104e; (Beauvais et al., 2005)) or against b1-3glucan (Biosupplies Australia, mouse IgG).
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or the endo-b1-3glucanases laminarinase A and Zymolyase T20. None of the enzyme treatments prevented germination. The only enzymatic activity common to both preparations was a1-3glucanase (Supplementary Table 2). a1-3Glucanase, lacking other glycosyl-hydrolases, was purified from the mutanase preparation (Mutanase–P). This enzyme prevented conidial aggregation (Fig. 2). The reversibility of the conidial aggregation was tested by incubation of the conidial aggregates with mutanase. After 30 min of enzymatic treatment, mutanase disintegrated all clumps (Fig. 3). In contrast, 100 mM HCl or 100 mM NaOH or proteinase K did not modify conidia aggregates. To confirm the importance of a1-3glucan in the conidial aggregation, cell wall polysaccharide fractions and commercial polysaccharides were tested in the aggregation assay. Among various tested polymers such as chitin, b1-3glucan, curdlan, lipogalactomannan and galactosaminogalactan, only mutan was able to inhibit the conidial aggregation in a way similar to the addition of mutanase (Fig. 2B). Optic microscopy showed that in presence of a patch of curdlan, conidial aggregates were formed and did not interact with the insoluble polysaccharide (Fig. 4). In contrast, no conidial clumps were observed in presence of mutan or AS-OxP, but swollen conidia adhered to these insoluble polysaccharides (Fig. 4). Acetolysis treatment of the mutan produced an insoluble fraction containing only a1-3glucan. This fraction inhibited similarly the conidial aggregation (data
3. Results In our experimental growth conditions, the aggregation of A. fumigatus conidia started 90 min after germination at 37 °C (Fig. 1). The size of the aggregates increased over time and was dependent on the conidial concentration and speed of orbital shaking. After 90 min, small clumps containing 10–100 conidia were observed but most of conidia remained loosely attached. After 3 h, large clumps with millions of conidia were seen and more than 95% of conidia were aggregated (Fig. 1). Since resting conidia do not agglutinate, the conidial agglutination could be due to the unmasking an underlying layer that leads to the appearance of new molecules at the conidial surface during germination. To determine if the removal of surface components during germination occurred, the agglutination of conidia from a pks mutant deficient in cell wall melanin and rodA mutant deficient in the rodlet hydrophobic layer on their cell surface were tested. The conidia of these mutants did not agglutinate until germination occurred (data not shown), indicating that the removal of the rodlet and melanin layers was not responsible for conidial agglutination. During the conidial swelling, the entire cell wall polysaccharide network must be remodelled. Specific polysaccharide hydrolases were added to the culture medium before inoculation. Only treatment with glucanex or mutanase prevented conidia aggregation (Fig. 2A). No effect was observed with chitinase, a-mannosidase
Fig. 2. Effect of glycosyl-hydrolases (A) and polysaccharides (B) on the conidial aggregation after 3 h of incubation at 37 °C under shaking (150 rpm). Glycosidases: Glx, 50 ll of glucanex (10 mg/ml); Zym, 50 ll of Zymolyase T20 (10 mg/ml); Lam-A, 10 ll of recombinant laminarinase A (2 mg/ml); Cht, 50 ll of recombinant chitinase A (0.5 mg/ml); 10 ll of mutanase, novo, crude preparation (12 mg/ml), P, 10 ll of purified preparation; JBAM, 50 ll of Jack bean a-mannosidase (6 mg/ml). Polysaccharides (0.2 mg/ml final concentration): Lam, laminarin, LGM, lipogalactomannan; GG, galactosaminogalactan. In panel B, mutanase treatment was used as a control for loss of agglutination.
Fig. 1. (A) Kinetics of conidial aggregation during germination in liquid Sabouraud medium over 3 h at 37 °C under shaking (150 rpm). (B) Microscopic visualisation of a conidial aggregate at 3 h. Bar = 30 lm.
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Fig. 3. Effect of protein degradation on conidial aggregates. Conidial aggregation was obtained after 3 h of incubation at 37 °C under shaking (150 rpm). Then conidial aggregates were treated with 10 ll of mutanase (Mut), 1 mg/ml of proteinase K at pH 7.5 (Prot-K), 100 mM NaOH (NaOH), 100 mM HCl (HCl) at 37 °C under shaking for 30 or 60 min. C, control without any treatment.
not shown). These data clearly indicate that a1-3glucans were essential to the aggregation of swollen conidia. Immunocytochemical analysis of the conidial cell wall showed the presence of a1-3glucan at the cell surface of swollen conidia (Fig. 5), indicating that a1-3glucans became exposed at the cell surface of conidia during conidial swelling. Self aggregation of conidia through cell surface a1-3glucan occurred, and the interaction was inhibited by the addition of a1-3glucanase or exogenous a1-3glucans. However, the mechanism of a1-3glucan recognition remains unknown. Acetolysis of the AS-OxP fraction during 72 h produced a1-3glucan-oligosaccharides of several sizes. Two fractions were easily separated by their water-solubility. The water soluble fraction, containing oligosaccharides of dp < 16, was then separated
by gel filtration into three fractions G15-I, G15-II and G15-III with a maximal dp 7–12, 6–10 and 4–8 respectively. The water-insoluble fraction contained dp > 10–40 glucose residues that is the limit of separation by HPAEC (Supplementary Fig. 1). Fig. 6 shows that the inhibition capacity of the a1-3glucan-oligosaccharides increased with the dp of the oligosaccharide. The water-insoluble fraction was able to inhibit conidial aggregation at a concentration of 62 lg/ml, similar to mutan. In contrast, the water soluble fractions, G15-I-III, were poorly inhibitory. The smaller oligosaccharides from soluble fractions were not inhibitors of the aggregation whereas larger soluble oligosaccharides inhibited at 2 mg/ml (Fig. 6). In our experimental conditions, the inhibition of swollen conidia aggregation requires a minimum dp > 12 and increases with an increase in the a1-3glucan chain size that becomes optimal with insoluble chains. Water-insoluble glucans obtained after acetolysis of AS-OxP fraction were conjugated to latex beads. Both b1-3glucan coated and a1-3glucan coated latex beads did not aggregate (Fig. 7). b1-3Glucan coated beads did not bind to mutan and so was used as negative control. Coated latex beads were inoculated with conidia or mutan or the AS-OxP fraction in Sabouraud medium in the same conditions of the aggregation assay. a1-3Glucan-coated beads bound to swollen conidia, showing that a1-3glucan chains were specifically interacting with the conidia cell surface (Fig. 7). The docking of a1-3glucan-coated beads to insoluble mutan and to AS-OxP fraction (Fig. 7) indicated that the a1-3glucan chains interacted without the requirement of other cell wall component, protein or polysaccharide.
Fig. 4. Phase contrast optic microscopy of swollen conidia in presence of insoluble polysaccharides (curdlan or mutan or AS-OxP). Conidia (107/ml) were incubated in Sabouraud medium at 37 °C under shaking (150 rpm) for 3 h in presence of 0.2 mg/ml of polysaccharide. Bar = 30 lm.
Fig. 5. Electron microscopy images of ultrathin sections of conidia of A. fumigatus after immunolabelling of the a1-3glucans with anti-a1,3glucan antibody of resting conidia (A) and 3 h germinated conidia (B) IL and OL, inner and outer layer of the conidial cell wall. Positive labelling is shown by gold particles.
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Fig. 6. Effect of the size of a1-3glucan chains on the conidial aggregation after 3 h of incubation at 37 °C under shaking (150 rpm). Fractions of a1-3glucan were obtained after acetolysis of cell wall a1-3glucan from A. fumigatus (their size is shown in Supplementary Fig. 1).
4. Discussion The aggregation of swollen fungal conidia has been described long ago (Metz and Kossen, 1977). However, this cell–cell interaction is not a common property of all fungal conidia after the breaking of dormancy. This phenomenon has been clearly observed in Aspergillus, Phanerochaete and Syncephalastrum species (Dute et al., 1989; Gerin et al., 1993; Grimm et al., 2004; Hobot and Gull, 1981; Tronchin et al., 1995). In the Phanerochaete genome, a homologue of the AGS (a1-3glucan synthase) gene has been observed. The genome of Syncephalastrum is not sequenced yet and no cell
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wall analysis has been done. Trichoderma reesii spores swell before the germination, but no aggregation has been observed (L. Hartl, personal communication) and no AGS gene has been found in its genome. When we inoculated conidia Penicillium chrysogenum, Penicillium verrucosum and Rhizopus oryzae strains in our assay at 25 °C, conidia of all strains swelled but only Penicillium species aggregated. These aggregations were inhibited by the presence of mutanase or mutan (data not shown). Two AGS genes are present in Penicillium species whereas none is found in Rhizopus strains. All species that have AGS in their genome and putatively a1-3glucan in their cell wall agglutinate whereas the two species that had no a1-3glucan synthase did not aggregate. a1-3Glucans are also involved in the anchoring of the capsule polysaccharide to the cell wall of Cryptococcus neoformans (Reese and Doering, 2003). Interestingly, yeast cells of Cryptococcus neoformans mutant deficient in capsule synthesis self-aggregated (Chang et al., 1996). Our data suggest that the absence of capsule unmasks cell wall a1-3glucan that become accessible and induces yeast agglutination. Three a-glucan synthase (AGS) genes have been described in A. fumigatus genome (Beauvais et al., 2005; Maubon et al., 2006). Conidia from all three single mutants aggregated as did the wild type strain (data not shown). None of the AGS gene is essential for the aggregation. ags2 and ags3 single mutants have the same amount of cell wall a-glucan as in wild type strain. A 50% reduction of aglucan has only been observed in ags1 mutant (Beauvais et al., 2005; Maubon et al., 2006). However, even this reduced amount of a1-3glucan did not modify the kinetic of conidial aggregation (data not shown). Mating, colony morphology changes, biofilm formation, fruiting body development and adhesion to host tissues require the interaction between fungal cells or fungal and host cells. Due to their external location, cell wall components, proteins and carbohydrates, are in contact with the external environment and are involved in specific recognition. Except for the well studied yeast mating interaction where a protein–protein recognition is involved, most of the cell adhesion processes are controlled by a protein-carbohydrate complex (Dranginis et al., 2007). Our experiments showed that a1-3glucan chains interacted between themselves and are respon-
Fig. 7. Fluorescence and phase contrast optic microscopy of a or b1-3glucan-coated latex beads in presence of swollen conidia or mutan or AS-OxP. 107 coated latex beads were incubated with 107 conidia or 200 lg of mutan or AS-OxP in 1 ml Sabouraud medium for 3 h at 37 °C, 150 rpm. Bar = 5 lm.
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sible for the agglutination of swollen conidia or to insoluble a1-3glucan coated beads without the intervention of any protein. Accordingly, the physical properties of insoluble a1-3glucan were responsible to the conidial aggregation. Even though a1-3glucan carbohydrate binding modules (CBM-24) have been previously described in filamentous fungi (Fuglsang et al., 2000), the lack of an effect by proteinase, 0.1 M NaOH, or 0.1 M HCl treatments suggested that no protein was involved in conidial agglutination. a1-3Glucan specific CBM have only been found to date at the C-terminus of a1-3glucanase. Interaction between linear b1-3glucan chains with the organisation of single and triple helix chain is well documented (Sletmoen and Stokke, 2008; Stone and Clarke, 1992). In spite of the cell surface exposure of b1-3glucan during conidial germination, this polysaccharide has no role in conidial recognition, suggesting that a1-3glucan–a1-3glucan interaction should be stronger than b1-3glucan interchain interaction. In A. fumigatus, a1-3glucan is the main component of conidia and mycelium cell wall. No covalently linkage to other cell wall component has been described. In A. fumigatus, a13glucans seem associated with melanin of the conidia cell wall (Maubon et al., 2006). a1-3Glucans are also found in the matrix of the aerial-grown colony (Beauvais et al., 2007). In vivo, a1-3glucan has been also observed in the matrix of biofilm during pulmonary aspergilloma, where hyphae remain strongly aggregated but not during invasive aspergillosis where hyphae were disseminated in the lung (Loussert et al., 2010), suggesting that a1-3glucans could be involved in the biofilm cohesion. Analysis of biophysical properties of the a1-3glucan and their interaction with other polysaccharides will lead not only to an understanding of the a1-3glucan–a13glucan interaction but also to a comprehension of the structural role inside the fungal cell wall. Acknowledgments We thank Doctor E. Rolides (Thessaloniki, Greece) for providing a Rhizopus oryzae strain and Professor R. Calderone (Georgetown University Medical center, Washington, USA) for reading of the manuscript.
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A new family of alpha1,3-glucanases with novel carbohydrate-binding domains. J. Biol. Chem. 275, 2009–2018. Gerin, P.A., Dufrene, Y., Bellon-Fontaine, M.N., Asther, M., Rouxhet, P.G., 1993. Surface properties of the conidiospores of Phanerochaete chrysosporium and their relevance to pellet formation. J. Bacteriol. 175, 5135–5144. Gersuk, G.M., Underhill, D.M., Zhu, L., Marr, K.A., 2006. Dectin-1 and TLRs permit macrophages to distinguish between different Aspergillus fumigatus cellular states. J. Immunol. 176, 3717–3724. Griffith, J., Mari, M., De Maziere, A., Reggiori, F., 2008. A cryosectioning procedure for the ultrastructural analysis and the immunogold labelling of yeast Saccharomyces cerevisiae. Traffic 9, 1060–1072. Grimm, L.H., Kelly, S., Hengstler, J., Gobel, A., Krull, R., Hempel, D.C., 2004. Kinetic studies on the aggregation of Aspergillus niger conidia. Biotechnol. Bioeng. 87, 213–218. Harris, S.D., Momany, M., 2004. Polarity in filamentous fungi: moving beyond the yeast paradigm. Fungal Genet. Biol. 41, 391–400. Hartland, R.P., Fontaine, T., Debeaupuis, J.P., Simenel, C., Delepierre, M., Latge, J.P., 1996. A novel beta-(1–3)-glucanosyltransferase from the cell wall of Aspergillus fumigatus. J. Biol. Chem. 271, 26843–26849. Hobot, J.A., Gull, K., 1981. Changes in the organisation of surface rodlets during germination of Syncephalastrum racemosum sporangiospores. Protoplasma 107, 339–343. Kelly, S., Grimm, L.H., Jonas, R., Hempel, D.C., Krull, R., 2006. Investigations of the morphogenesis of filamentous microorganisms. Eng. Life Sci. 6, 475–480. Lamarre, C., Sokol, S., Debeaupuis, J.P., Henry, C., Lacroix, C., Glaser, P., Coppee, J.Y., Francois, J.M., Latge, J.P., 2008. Transcriptomic analysis of the exit from dormancy of Aspergillus fumigatus conidia. BMC Genom. 9, 417–431. Lin, P.J., Grimm, L.H., Wulkow, M., Hempel, D.C., Krull, R., 2008. Population balance modeling of the conidial aggregation of Aspergillus niger. Biotechnol. Bioeng. 99, 341–350. Loussert, C., Schmitt, C., Prevost, M.C., Balloy, V., Fadel, E., Philippe, B., KauffmannLacroix, C., Latgé, J.P., Beauvais, A., 2010. In vivo biofilm composition of Aspergillus fumigatus. Cell. Microbiol. 12, 405–410. Luther, K., Torosantucci, A., Brakhage, A.A., Heesemann, J., Ebel, F., 2007. Phagocytosis of Aspergillus fumigatus conidia by murine macrophages involves recognition by the dectin-1 beta-glucan receptor and toll-like receptor 2. Cell Microbiol. 9, 368–381. Martin-Cuadrado, A.B., Encinar del Dedo, J., de Medina-Redondo, M., Fontaine, T., del Rey, F., Latge, J.P., Vazquez de Aldana, C.R., 2008. The Schizosaccharomyces pombe endo-1,3-beta-glucanase Eng1 contains a novel carbohydrate binding module required for septum localization. Mol. Microbiol. 69, 188–200. Maubon, D., Park, S., Tanguy, M., Huerre, M., Schmitt, C., Prevost, M.C., Perlin, D.S., Latge, J.P., Beauvais, A., 2006. AGS3, an alpha(1–3)glucan synthase gene family member of Aspergillus fumigatus, modulates mycelium growth in the lung of experimentally infected mice. Fungal Genet. Biol. 43, 366–375. Metz, B., Kossen, N.W.F., 1977. The growth of molds in the form of pellets – a literature review. Biotechnol. Bioeng. 19, 781–799. Papagianni, M., 2004. Fungal morphology and metabolite production in submerged mycelial processes. Biotechnol. Adv. 22, 189–259. Reese, A.J., Doering, T.L., 2003. Cell wall alpha-1,3-glucan is required to anchor the Cryptococcus neoformans capsule. Mol. Microbiol. 50, 1401–1409. Rohde, M., Schwienbacher, M., Nikolaus, T., Heesemann, J., Ebel, F., 2002. Detection of early phase specific surface appendages during germination of Aspergillus fumigatus conidia. FEMS Microbiol. Lett. 206, 99–105. Ryoo, D.H., Choi, C.S., 1999. Surface thermodynamics of pellet formation in Aspergillus niger. Biotechnol. Lett. 21, 97–100. Sletmoen, M., Stokke, B.T., 2008. Higher order structure of (1,3)-beta-D-glucans and its influence on their biological activities and complexation abilities. Biopolymers 89, 310–321. Stone, B.A., Clarke, A.E., 1992. Structural Characterisation and Physical Chemistry of (1–3)-b-Glucans. Chemistry and Biology of (1–3)-b-Glucans. La Trobe University Press, Victoria. Sugawara, T., Sato, M., Takagi, T., Kamasaki, T., Ohno, N., Osumi, M., 2003. In situ localization of cell wall alpha-1,3-glucan in the fission yeast Schizosaccharomyces pombe. J. Electron. Microsc. (Tokyo) 52, 237–242. Tronchin, G., Bouchara, J.P., Ferron, M., Larcher, G., Chabasse, D., 1995. Cell surface properties of Aspergillus fumigatus conidia: correlation between adherence, agglutination, and rearrangements of the cell wall. Can. J. Microbiol. 41, 714–721. 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Cell wall a1-3glucans induce the aggregation of germinating conidia of Aspergillus fumigatus Thierry Fontaine a,*, Anne Beauvais a, Céline Loussert b, Benoît Thevenard a,1, Claus. C. Fulgsang c, Naohito Ohno d, Cécile Clavaud a, Marie-Christine Prevost b, Jean-Paul Latgé a a
Unité des Aspergillus, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris cedex 15, France Plateforme de Microscopie Électronique, Institut Pasteur, 25 rue du Docteur Roux, 75724 Paris cedex 15, France Novozymes A/S Bagwaerd DK-2880, Denmark d School of Pharmacy, Tokyo University of Pharmacy and Life science, Tokyo 192-0392, Japan b c
a r t i c l e
i n f o
Article history: Received 8 March 2010 Accepted 27 April 2010 Available online 4 May 2010 Keywords: Aspergillus fumigatus Conidial germination a1-3Glucan Cell wall
a b s t r a c t The germination of Aspergillus fumigatus conidia can be divided into four stages: breaking of dormancy, isotropic swelling, establishment of cell polarity, and formation of a germ tube. Swelling of conidia is associated in liquid medium with a multi-cellular aggregation that produced large clumps of conidia. Conidial aggregation can be specifically prevented by the addition of a1-3glucanase. Swollen conidia specifically adhere to insoluble a1-3glucan chains. Electron microscopy studies showed that cell wall a1-3glucan chains became exposed at the cell surface during the swelling. These results demonstrate that cell wall a1-3glucans play an essential role in the aggregation between swollen conidia. Experiments with a1-3glucan coated latex beads show that a1-3glucan chains interacted between them without the requirement of any other cell wall component suggesting that biophysical properties of a1-3glucans are solely responsible for conidial aggregation. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Aspergillus species produce small hydrophobic conidia that are dispersed into the air. Under favourable nutritional conditions, conidia germinate to colonise new environments. Conidial germination can be divided into four steps: breaking of the dormancy, isotropic swelling, establishment of cell polarity and formation of a germ tube (Barhoom and Sharon, 2004; d’Enfert, 1997; Harris and Momany, 2004). In liquid medium, conidial germination of Aspergillus fumigatus conidia was associated with conidial agglutination (Lamarre et al., 2008), that has been described in a number of filamentous fungi many years ago (Metz and Kossen, 1977). Conidial agglutination does not influence the total germination, but increases the size of the pellets produced under shake conditions and affects the use of such filamentous fungi in biotechnology (Kelly et al., 2006; Papagianni, 2004). In Aspergillus niger, a mathe-
Abbreviations: DMSO, dimethylsulfoxide; AS-OxP, periodate oxided alkalisoluble cell wall fraction of A. fumigatus that contains only a1-3glucans; HPAEC, high performance anion exchange chromatography; LGM, lipogalactomannan. * Corresponding author. E-mail address: [email protected] (T. Fontaine). 1 Present address: UR INRA No. 0477, Biochimie bactérienne, Domaine de Vilvert, 78332 Jouy en Josas, France. 1087-1845/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.fgb.2010.04.006
matic model suggests that conidial aggregation corresponds to an agglomeration step but not a tight intergrowth of particles (Lin et al., 2008), suggesting a cell surface recognition. However, the cell surface molecules involved in this cellular interaction remain unknown. The conidial cell wall is composed of two layers: an external hydrophobic layer containing melanin and rodlet proteins and an internal layer, electron translucent, containing polysaccharides such as a and b-glucans, chitin/chitosan and galactomannan (Maubon et al., 2006). During germination, swelling of conidia requires cell wall remodelling. Indeed, during the isodiametric growth, the conidial surface is dramatically changed. Scanning electron microscopy and by atomic force microscopy have demonstrated that the outer rodlet/melanin layer of the resting conidia is progressively lost and the cell surface layer is changed into a layer of amorphous material unmasking b1-3glucan on the surface of the conidium (Dague et al., 2008; Gersuk et al., 2006; Luther et al., 2007; Rohde et al., 2002). Earlier studies have suggested that glucan exposure or changes in electrical charges and hydrophobicity could be responsible for the agglutination process but no experimental proof was given (Dynesen and Nielsen, 2003; Ryoo and Choi, 1999; Tronchin et al., 1995). In this study we showed that cell wall a1-3glucans, unmasked during germination, are responsible for the aggregation of swollen conidia of A. fumigatus.
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2. Materials and methods
2.4. Isolation of A. fumigatus polysaccharides
2.1. Strains, media, enzymes and chemical compounds
The lipogalactomannan was prepared from mycelium membrane of A. fumigatus (Costachel et al., 2005). The galactosaminogalactan is extracted from the Brian culture medium of A. fumigatus and precipitated with 2.5 vol. of ethanol at 4 °C overnight. After centrifugation (5000g, 10 min), the pellet was washed with 150 mM NaCl. The galactosaminogalactan was extracted by 8 M urea and then dialysed against water and freeze-dried (Delangle, unpublished results). Alkali-insoluble (AI) and alkali-soluble (AS) fractions were prepared from the mycelial cell wall of A. fumigatus as previously described (Fontaine et al., 2000). Cell wall a1-3glucan containing the AS fraction was purified by periodate oxidation. The AS fraction was treated with 100 mM NaIO4 at 4 °C for 5 days in the dark. After the addition of ethylene glycol to destroy the excess of reagent, cell wall fractions were dialysed, reduced with BH4Na, and hydrolysed by Smith degradation (10% acetic acid 100 °C, 1 h). After dialysis against water and freeze-drying, a1-3glucan-oligosaccharides were produced by acetolysis. One gram of periodate-treated AS fraction (AS-OxP) was acetolysed with 100 ml of acetic acid/acetic anhydride/sulphuric acid solution (10/10/1, v/v/v) for 3 days at room temperature under magnetic stirring. The reaction was stopped in a water–ice bath by the addition of pyridine until a neutral pH was achieved. Peracetylated products were extensively dialysed against water at 4 °C and freeze-dried. De-O-acetylation was done by NaOH 0.1 M at room temperature. After centrifugation (5000g for 10 min), the supernatant was neutralised by the addition of acetic acid and fractionated by gel filtration on a G15-Sephadex column (GE Healthcare, 90 130 cm) equilibrated in 0.2% acetic acid and eluted at the flow rate of 7.5 ml/h. Insoluble material was removed by centrifugation, dialysed against water and freeze-dried. The size of acetolysed oligosaccharides was estimated by anion exchange chromatography equipped with a pulsed electrochemical detector (model ICS-3000, Dionex) with a CarboPAC PA-200 column (4.5 250 mm, Dionex) using the following conditions: flow rate 0.35 ml/min; solvent A, NaOH 50 mM; solvent B, NaOAc 500 mM in NaOH 50 mM; gradient initial time to 2 min isocratic elution with 98% A, 2% B; 2–15 min: 2–35% B; 15–35 min 35–100% B; 35–40 min isocratic elution with 100% B (Supplementary Fig. 1).
The fungal strains used in this study, A. fumigatus, Penicillium and Rhizopus species are presented in the Supplementary Table 1. Strains were maintained on 2% malt agar tubes. Mutan (waterinsoluble a-glucan containing mainly a1-3linkages) and mutanase, an a1-3glucanase from Trichoderma harzianum, were obtained as described (Fuglsang et al., 2000). Chitin and laminarin (soluble b1-3glucan) from Laminaria digitata were purchased from Sigma. Curdlan (insoluble b1-3glucan) is a kind gift from Dr. Hidemitsu Kobayashi. Mutanase was purified as previously described (Dekker et al., 2004). Zymolyase T20 from Arthrobacter luteus was purchased from ICN. Jack bean a-mannosidase was purchased from Sigma. Glucanex, a b1-3glucanase cocktail, was purchased from Novo Nordisk Ferment Ltd., (Dittinger, Switzerland). Chitinase A from Serratia macerans, recombinant in Escherichia coli, was a kind gift from Dr. C. Vorgias (Vorgias et al., 1993). Recombinant laminarinase A protein isolated from Thermotoga neapolitana, and expressed in E. coli, a kind gift of Dr. V. Zverlov (Institute of Molecular Genetics, Russian academy of sciences, Moscow), was purified as previously described (Zverlov et al., 1997). Proteinase K was purchased from Roche. Fungal orthologous of AGS genes have been search in the NCBI database (http://www.ncbi.nlm.nih. gov/sutils/genom_table.cgi?organism=fungi). 2.2. Enzyme assays Glycosyl hydrolase activity was quantified in 150 mM sodium acetate pH 5.5. p-Nitrophenyl-a-D-mannoside (Sigma) was used as a substrate for the Jack bean a-mannosidase activity (Bischoff et al., 1990). b1-3Glucanase and a1-3glucanase activities were determined with reduced laminarin or reduced mutan, respectively and quantified with the p-hydroxybenzoic acid hydrazide reagent (Hartland et al., 1996). Chitinase activity was determined using the fluorogenic substrates of methyl-umbeliferiyl-chitobiose (4-MU-(GlcNAc)2, Sigma) at the final concentration of 75 lM. The amount of 4-MU released from the assay reaction was determined at 320 nm excitation and 448 nm emission using a spectrofluorometer. Corresponding activities were summarised in Supplementary Table 2. 2.3. Conidial aggregation assay Conidia were recovered from slants using an aqueous 0.05% Tween 20 solution to obtain a homogeneous suspension. Aggregation assays were done in 24 well plates (TPP, Trasadingen, Switzerland) in 1 ml Sabouraud liquid medium (2% glucose and 1% mycopeptone, Oxoid) containing 0.05% Tween 20 and agitated at 150 rpm at 37 °C for 30 min to 3 h. The presence of 0.05% Tween 20 was required to maintain conidia in suspension in the liquid medium. Agglutination was monitored over time by visual examination. Putative inhibitors of aggregation were added to Sabouraud medium before the inoculation of conidia. Mutan and curdlan are two water-insoluble glucans. To obtain a homogeneous suspension, mutan was first solubilised in DMSO (5 mg/ml) and then precipitated by dilution in water (5 vol.). The precipitate was recovered by centrifugation (5000g, 10 min), washed with water and kept at 4 °C at 5 mg/ml. Curdlan was suspended at 5 mg/ml in water, heated at 65 °C for 30 min and stored at 4 °C. The conidial aggregation assays with Penicillium and Rhizopus strains were done at 25 °C. Conidial aggregate obtained after 3 h of germination were tested for their sensitivity to mutanase, proteinase K, and acidic and basic treatments (100 mM NaOH and 100 mM HCl) that unfold proteins.
2.5. Electron microscopy Resting and 3 h germinating conidia were fixed in 2% paraformaldehyde, 0.2% glutaraldehyde in 0.1 M Sorensen buffer at room temperature for 2 h then overnight at 4 °C. After fixation, free aldehydes were blocked by incubation in 50 mM NH4Cl in PBS for 15 min. Then samples were embedded in 12% gelatin. After solidification, samples were cut into small blocks and then transferred to 2.1 M sucrose in 0.1 M Sorensen buffer for 48 h at 4 °C. Finally, blocks were mounted on pins and frozen in liquid nitrogen. Cryosectioning was done using an UltraCut EM FC6 (Leica, Vienna, Austria) at 110 °C, and 50 nm sections were picked up with 0.8% methylcellulose, 1.26 M sucrose in 0.06 M Sorensen buffer. Immunolabelling of sections on copper grids was done as previously described (Griffith et al., 2008) using a polyclonal antibody directed against a1-3 glucan (Sugawara et al., 2003). The primary antibody was diluted 1:50, then treated with an anti-rabbit F (ab0 )2 goat antibody (diluted 1:25) coupled to 10 nm colloidal gold (BBI) (Beauvais et al., 2007). Sections were observed under 80 kV in a Jeol transmission electron microscope (Jem 1010). 2.6. Coating of glucan to latex beads Twenty milligrams of water-insoluble fraction of acetolysed and borohydride-reduced AS fraction were incubated in 1 ml of
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50 mM IO4Na for 30 min on ice. After addition of 100 ll of glycerol, oxidised glucans were desalted by centrifugation and washed with water. Thirty-four milligrams of biotin-hydrazide (Pierce) were dissolved in 3 ml of DMSO, added to the glucan pellet and incubated for 3 h at room temperature. After the addition of 3 ml of 0.2 M NaOAc pH 5.5, the mixture was left overnight at room temperature, then dialysed against water and freeze-dried. Two milligrams of biotinylated a1-3glucan was dissolved in 500 ll of DMSO, then diluted with 500 ll of water and incubated with 50 ll of streptavidin-activated latex beads (Sigma, 1 lm, 1010 beads/ml, red labelled) for 90 min at room temperature. After addition of 500 ll of water, excess of biotinylated glucans was removed by centrifugation (10 min, 8000g). Latex beads were then washed once with 30% of DMSO and twice with water and kept at 4 °C. b1-3Glucan coated latex beads were used as negative control in the aggregation assay. b1-3Glucan chains were obtained by acetolysis as previously described (Martin-Cuadrado et al., 2008). Biotinylation and coupling to streptavidin-activated latex beads were done as described for a1-3glucans. The coating of glucan on latex beads was confirmed by immunofluorescence using specific antibodies against a1-3glucan (Sigma, mouse IgM MOPCk 104e; (Beauvais et al., 2005)) or against b1-3glucan (Biosupplies Australia, mouse IgG).
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or the endo-b1-3glucanases laminarinase A and Zymolyase T20. None of the enzyme treatments prevented germination. The only enzymatic activity common to both preparations was a1-3glucanase (Supplementary Table 2). a1-3Glucanase, lacking other glycosyl-hydrolases, was purified from the mutanase preparation (Mutanase–P). This enzyme prevented conidial aggregation (Fig. 2). The reversibility of the conidial aggregation was tested by incubation of the conidial aggregates with mutanase. After 30 min of enzymatic treatment, mutanase disintegrated all clumps (Fig. 3). In contrast, 100 mM HCl or 100 mM NaOH or proteinase K did not modify conidia aggregates. To confirm the importance of a1-3glucan in the conidial aggregation, cell wall polysaccharide fractions and commercial polysaccharides were tested in the aggregation assay. Among various tested polymers such as chitin, b1-3glucan, curdlan, lipogalactomannan and galactosaminogalactan, only mutan was able to inhibit the conidial aggregation in a way similar to the addition of mutanase (Fig. 2B). Optic microscopy showed that in presence of a patch of curdlan, conidial aggregates were formed and did not interact with the insoluble polysaccharide (Fig. 4). In contrast, no conidial clumps were observed in presence of mutan or AS-OxP, but swollen conidia adhered to these insoluble polysaccharides (Fig. 4). Acetolysis treatment of the mutan produced an insoluble fraction containing only a1-3glucan. This fraction inhibited similarly the conidial aggregation (data
3. Results In our experimental growth conditions, the aggregation of A. fumigatus conidia started 90 min after germination at 37 °C (Fig. 1). The size of the aggregates increased over time and was dependent on the conidial concentration and speed of orbital shaking. After 90 min, small clumps containing 10–100 conidia were observed but most of conidia remained loosely attached. After 3 h, large clumps with millions of conidia were seen and more than 95% of conidia were aggregated (Fig. 1). Since resting conidia do not agglutinate, the conidial agglutination could be due to the unmasking an underlying layer that leads to the appearance of new molecules at the conidial surface during germination. To determine if the removal of surface components during germination occurred, the agglutination of conidia from a pks mutant deficient in cell wall melanin and rodA mutant deficient in the rodlet hydrophobic layer on their cell surface were tested. The conidia of these mutants did not agglutinate until germination occurred (data not shown), indicating that the removal of the rodlet and melanin layers was not responsible for conidial agglutination. During the conidial swelling, the entire cell wall polysaccharide network must be remodelled. Specific polysaccharide hydrolases were added to the culture medium before inoculation. Only treatment with glucanex or mutanase prevented conidia aggregation (Fig. 2A). No effect was observed with chitinase, a-mannosidase
Fig. 2. Effect of glycosyl-hydrolases (A) and polysaccharides (B) on the conidial aggregation after 3 h of incubation at 37 °C under shaking (150 rpm). Glycosidases: Glx, 50 ll of glucanex (10 mg/ml); Zym, 50 ll of Zymolyase T20 (10 mg/ml); Lam-A, 10 ll of recombinant laminarinase A (2 mg/ml); Cht, 50 ll of recombinant chitinase A (0.5 mg/ml); 10 ll of mutanase, novo, crude preparation (12 mg/ml), P, 10 ll of purified preparation; JBAM, 50 ll of Jack bean a-mannosidase (6 mg/ml). Polysaccharides (0.2 mg/ml final concentration): Lam, laminarin, LGM, lipogalactomannan; GG, galactosaminogalactan. In panel B, mutanase treatment was used as a control for loss of agglutination.
Fig. 1. (A) Kinetics of conidial aggregation during germination in liquid Sabouraud medium over 3 h at 37 °C under shaking (150 rpm). (B) Microscopic visualisation of a conidial aggregate at 3 h. Bar = 30 lm.
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Fig. 3. Effect of protein degradation on conidial aggregates. Conidial aggregation was obtained after 3 h of incubation at 37 °C under shaking (150 rpm). Then conidial aggregates were treated with 10 ll of mutanase (Mut), 1 mg/ml of proteinase K at pH 7.5 (Prot-K), 100 mM NaOH (NaOH), 100 mM HCl (HCl) at 37 °C under shaking for 30 or 60 min. C, control without any treatment.
not shown). These data clearly indicate that a1-3glucans were essential to the aggregation of swollen conidia. Immunocytochemical analysis of the conidial cell wall showed the presence of a1-3glucan at the cell surface of swollen conidia (Fig. 5), indicating that a1-3glucans became exposed at the cell surface of conidia during conidial swelling. Self aggregation of conidia through cell surface a1-3glucan occurred, and the interaction was inhibited by the addition of a1-3glucanase or exogenous a1-3glucans. However, the mechanism of a1-3glucan recognition remains unknown. Acetolysis of the AS-OxP fraction during 72 h produced a1-3glucan-oligosaccharides of several sizes. Two fractions were easily separated by their water-solubility. The water soluble fraction, containing oligosaccharides of dp < 16, was then separated
by gel filtration into three fractions G15-I, G15-II and G15-III with a maximal dp 7–12, 6–10 and 4–8 respectively. The water-insoluble fraction contained dp > 10–40 glucose residues that is the limit of separation by HPAEC (Supplementary Fig. 1). Fig. 6 shows that the inhibition capacity of the a1-3glucan-oligosaccharides increased with the dp of the oligosaccharide. The water-insoluble fraction was able to inhibit conidial aggregation at a concentration of 62 lg/ml, similar to mutan. In contrast, the water soluble fractions, G15-I-III, were poorly inhibitory. The smaller oligosaccharides from soluble fractions were not inhibitors of the aggregation whereas larger soluble oligosaccharides inhibited at 2 mg/ml (Fig. 6). In our experimental conditions, the inhibition of swollen conidia aggregation requires a minimum dp > 12 and increases with an increase in the a1-3glucan chain size that becomes optimal with insoluble chains. Water-insoluble glucans obtained after acetolysis of AS-OxP fraction were conjugated to latex beads. Both b1-3glucan coated and a1-3glucan coated latex beads did not aggregate (Fig. 7). b1-3Glucan coated beads did not bind to mutan and so was used as negative control. Coated latex beads were inoculated with conidia or mutan or the AS-OxP fraction in Sabouraud medium in the same conditions of the aggregation assay. a1-3Glucan-coated beads bound to swollen conidia, showing that a1-3glucan chains were specifically interacting with the conidia cell surface (Fig. 7). The docking of a1-3glucan-coated beads to insoluble mutan and to AS-OxP fraction (Fig. 7) indicated that the a1-3glucan chains interacted without the requirement of other cell wall component, protein or polysaccharide.
Fig. 4. Phase contrast optic microscopy of swollen conidia in presence of insoluble polysaccharides (curdlan or mutan or AS-OxP). Conidia (107/ml) were incubated in Sabouraud medium at 37 °C under shaking (150 rpm) for 3 h in presence of 0.2 mg/ml of polysaccharide. Bar = 30 lm.
Fig. 5. Electron microscopy images of ultrathin sections of conidia of A. fumigatus after immunolabelling of the a1-3glucans with anti-a1,3glucan antibody of resting conidia (A) and 3 h germinated conidia (B) IL and OL, inner and outer layer of the conidial cell wall. Positive labelling is shown by gold particles.
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Fig. 6. Effect of the size of a1-3glucan chains on the conidial aggregation after 3 h of incubation at 37 °C under shaking (150 rpm). Fractions of a1-3glucan were obtained after acetolysis of cell wall a1-3glucan from A. fumigatus (their size is shown in Supplementary Fig. 1).
4. Discussion The aggregation of swollen fungal conidia has been described long ago (Metz and Kossen, 1977). However, this cell–cell interaction is not a common property of all fungal conidia after the breaking of dormancy. This phenomenon has been clearly observed in Aspergillus, Phanerochaete and Syncephalastrum species (Dute et al., 1989; Gerin et al., 1993; Grimm et al., 2004; Hobot and Gull, 1981; Tronchin et al., 1995). In the Phanerochaete genome, a homologue of the AGS (a1-3glucan synthase) gene has been observed. The genome of Syncephalastrum is not sequenced yet and no cell
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wall analysis has been done. Trichoderma reesii spores swell before the germination, but no aggregation has been observed (L. Hartl, personal communication) and no AGS gene has been found in its genome. When we inoculated conidia Penicillium chrysogenum, Penicillium verrucosum and Rhizopus oryzae strains in our assay at 25 °C, conidia of all strains swelled but only Penicillium species aggregated. These aggregations were inhibited by the presence of mutanase or mutan (data not shown). Two AGS genes are present in Penicillium species whereas none is found in Rhizopus strains. All species that have AGS in their genome and putatively a1-3glucan in their cell wall agglutinate whereas the two species that had no a1-3glucan synthase did not aggregate. a1-3Glucans are also involved in the anchoring of the capsule polysaccharide to the cell wall of Cryptococcus neoformans (Reese and Doering, 2003). Interestingly, yeast cells of Cryptococcus neoformans mutant deficient in capsule synthesis self-aggregated (Chang et al., 1996). Our data suggest that the absence of capsule unmasks cell wall a1-3glucan that become accessible and induces yeast agglutination. Three a-glucan synthase (AGS) genes have been described in A. fumigatus genome (Beauvais et al., 2005; Maubon et al., 2006). Conidia from all three single mutants aggregated as did the wild type strain (data not shown). None of the AGS gene is essential for the aggregation. ags2 and ags3 single mutants have the same amount of cell wall a-glucan as in wild type strain. A 50% reduction of aglucan has only been observed in ags1 mutant (Beauvais et al., 2005; Maubon et al., 2006). However, even this reduced amount of a1-3glucan did not modify the kinetic of conidial aggregation (data not shown). Mating, colony morphology changes, biofilm formation, fruiting body development and adhesion to host tissues require the interaction between fungal cells or fungal and host cells. Due to their external location, cell wall components, proteins and carbohydrates, are in contact with the external environment and are involved in specific recognition. Except for the well studied yeast mating interaction where a protein–protein recognition is involved, most of the cell adhesion processes are controlled by a protein-carbohydrate complex (Dranginis et al., 2007). Our experiments showed that a1-3glucan chains interacted between themselves and are respon-
Fig. 7. Fluorescence and phase contrast optic microscopy of a or b1-3glucan-coated latex beads in presence of swollen conidia or mutan or AS-OxP. 107 coated latex beads were incubated with 107 conidia or 200 lg of mutan or AS-OxP in 1 ml Sabouraud medium for 3 h at 37 °C, 150 rpm. Bar = 5 lm.
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sible for the agglutination of swollen conidia or to insoluble a1-3glucan coated beads without the intervention of any protein. Accordingly, the physical properties of insoluble a1-3glucan were responsible to the conidial aggregation. Even though a1-3glucan carbohydrate binding modules (CBM-24) have been previously described in filamentous fungi (Fuglsang et al., 2000), the lack of an effect by proteinase, 0.1 M NaOH, or 0.1 M HCl treatments suggested that no protein was involved in conidial agglutination. a1-3Glucan specific CBM have only been found to date at the C-terminus of a1-3glucanase. Interaction between linear b1-3glucan chains with the organisation of single and triple helix chain is well documented (Sletmoen and Stokke, 2008; Stone and Clarke, 1992). In spite of the cell surface exposure of b1-3glucan during conidial germination, this polysaccharide has no role in conidial recognition, suggesting that a1-3glucan–a1-3glucan interaction should be stronger than b1-3glucan interchain interaction. In A. fumigatus, a1-3glucan is the main component of conidia and mycelium cell wall. No covalently linkage to other cell wall component has been described. In A. fumigatus, a13glucans seem associated with melanin of the conidia cell wall (Maubon et al., 2006). a1-3Glucans are also found in the matrix of the aerial-grown colony (Beauvais et al., 2007). In vivo, a1-3glucan has been also observed in the matrix of biofilm during pulmonary aspergilloma, where hyphae remain strongly aggregated but not during invasive aspergillosis where hyphae were disseminated in the lung (Loussert et al., 2010), suggesting that a1-3glucans could be involved in the biofilm cohesion. Analysis of biophysical properties of the a1-3glucan and their interaction with other polysaccharides will lead not only to an understanding of the a1-3glucan–a13glucan interaction but also to a comprehension of the structural role inside the fungal cell wall. Acknowledgments We thank Doctor E. Rolides (Thessaloniki, Greece) for providing a Rhizopus oryzae strain and Professor R. Calderone (Georgetown University Medical center, Washington, USA) for reading of the manuscript.
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