Purification and Partial Characterization of a 32-Kilodalton Sialic Acid-Specific Lectin from Aspergillus fumigatus

Abstract

Adherence of the opportunistic fungus Aspergillus fumigatus to the extracellular matrix components is considered a crucial step in the establishment of the infection. Given the high carbohydrate content of these glycoproteins and the role of carbohydrate-protein interactions in numerous adherence processes, the presence of a lectin in A. fumigatus was investigated. Different fungal extracts obtained by sonication or grinding in liquid nitrogen from resting or swollen conidia, as well as from germ tubes and mycelium, were tested by hemagglutination assays using rabbit erythrocytes. A lectin activity was recovered in all the extracts tested. However, sonication of resting conidia resulted in the highest specific activity. Purification of the lectin was achieved by gel filtration followed by ion-exchange and hydrophobic-interaction chromatographies. Analysis of the purified lectin by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed an apparent molecular mass of 32 kDa, which is similar to that of the alkaline protease already identified from different strains of A. fumigatus. However, as evidenced by the use of an alkaline protease-deficient mutant, the two activities were supported by distinct proteins. In addition, hemagglutination inhibition experiments using different saccharides and glycoproteins demonstrated the specificity of the lectin for sialic acid residues. Together these results suggest that this lectin may contribute to the attachment of conidia to the extracellular matrix components through the recognition of the numerous terminal sialic acid residues of their carbohydrate chains.

Usually a saprophyte, Aspergillus fumigatus grows and sporulates abundantly in a humid environment on decaying organic matter, leading to highly dispersible conidia. Therefore, this pathogen elicits by inhalation of its airborne conidia a wide spectrum of diseases, ranging from localized infections to invasive pulmonary aspergillosis. With the development of organ and bone marrow transplantations, the prevalence of this last clinical form has increased markedly during the past decades, and this fungus has become a major hospital pathogen (20, 27).

During the past few years, several groups have focused their research on the identification of cell wall factors associated with the pathogenicity of A. fumigatus. Indeed, the cell wall, located at the interface between the fungus and the host cells, plays a major role in the establishment of the infection. Thus, it has been demonstrated that the conidium's pigmentation contributes to fungal resistance against the host defense mechanisms and thus significantly influences the virulence of the fungus in a murine model (16, 22). Likewise, there is now accumulating evidence that adherence of the inhaled conidia to damaged epithelium, which is the initial event of the infection, is mediated by specific interactions of the fungus with some extracellular matrix components, i.e., type I and type IV collagens, fibrinogen, fibronectin, and basement membrane laminin (3, 6, 11, 31, 39). Some of the fungal receptors involved in these interactions have been identified at the molecular level. Attachment of conidia to fibronectin appeared to be mediated by two polypeptides of 23 and 30 kDa (31), whereas laminin was specifically recognized by a 72-kDa cell wall receptor (41). In addition, a low-molecular-mass hydrophobic protein of the conidial wall has been characterized, but it does not seem to be involved in adherence to extracellular matrix components or to alveolar type II pneumocytes (30, 37).

Another type of interaction which could provide a mechanism for the attachment of the fungus to the host cells may be accomplished by the specific recognition of glycoconjugates on the epithelial cell membrane by lectins present at the surface of the cell wall. Lectins in bacteria and protozoa have been extensively studied (18, 24, 26, 33, 36), but only a few lectins in human pathogenic fungi including Candida albicans (5, 10), Candida glabrata (9), and Histoplasma capsulatum (28), as well as some dermatophytes and related keratinolytic fungi (2, 7), have been described. In A. fumigatus, a 32-kDa fucose-specific lectin, concentrated in conidia rather than in mycelium, has been identified by Ishimaru et al. (T. Ishimaru, E. M. Bernard, S. Tamada, and D. Armstrong, Letter, Clin. Infect. Dis. 23:898, 1996). Nevertheless, further studies are needed to characterize this lectin in A. fumigatus.

Keeping in view the potential role of lectins in the pathogenesis of microorganisms, we searched for molecules supporting hemagglutination activity in A. fumigatus. In the present paper, we describe the purification and the characterization of a 32-kDa lectin with specificity towards sialic acid-containing glycoconjugates.

MATERIALS AND METHODS

Organisms and culture conditions.

A. fumigatus CBS 113.26 (Centraalbureau voor Schimmelcultures, Baarn, The Netherlands) was grown on yeast extract-peptone-dextrose agar at 37°C, and conidia were obtained from 5-day-old cultures by scraping the aerial mycelium in distilled water as described earlier (40). They were then pelleted by centrifugation (1,200 × g, 3 min) and resuspended in distilled water, and the absorbance at 620 nm of the obtained suspension was adjusted to 0.6 (approximately 10⁸ conidia/ml).

Swollen conidia and germ tubes were obtained by inoculating 1.5 ml of the conidial suspension in petri dishes containing 15 ml of medium 199 (Sigma Laboratories) buffered at pH 7.6 as reported previously (40). After incubation at 37°C, the fungal elements were pelleted by centrifugation and resuspended in phosphate-buffered saline (PBS) (0.15 M, pH 7.2). For the production of mycelium, the strain was grown in yeast extract-peptone-dextrose broth. Flasks (2 liters) containing 1 liter of medium were inoculated with approximately 10⁷ conidia per ml and incubated for 5 days at 37°C. The mycelium was harvested by filtration, washed with PBS, and briefly pressed dry.

An alkaline protease (ALP)-deficient mutant of A. fumigatus (ALP-nonproducing Δ18) kindly given by S. Paris (Pasteur Institute, Paris, France) was also used for some experiments.

Preparation of erythrocyte suspension.

Rabbit blood was collected in tubes containing EDTA and centrifuged at 3,000 × g for 5 min at 4°C. Erythrocyte suspensions were freshly prepared by washing the erythrocytes three times in PBS containing 10 mM EDTA, followed by two other washes with 20 volumes of 75 mM phosphate buffer (pH 7.2) containing 75 mM NaCl. Erythrocytes were then resuspended in PBS as a 10% (vol/vol) suspension, fixed with 3% formaldehyde in phosphate buffer for 18 h at 37°C, washed extensively in PBS, and stored at 4°C until used.

Preparation of fungal extracts.

Fungal extracts were prepared from the different morphological phases of the fungus (resting or swollen conidia, germ tubes, and mycelium), and two distinct extraction procedures were used. For the first one, the fungal elements were ground in a mortar with liquid nitrogen. The frozen mycelial powder was resuspended in an equal volume of PBS and centrifuged at 50,000 × g for 30 min. The supernatant was collected and is hereafter referred to as fungal extract I. For the other procedure, the fungal elements were sonicated four times at 200 W for 30 s (Sonifier Cell Disruptor; Branson Ultrasonics Co.) in an ice bath. After centrifugation at 50,000 × g for 30 min, the supernatant was collected and is hereafter referred to as fungal extract II. The protein concentration of each extract was determined by the method of Bradford (4) with bovine serum albumin as a standard.

In some cases, fungal extracts were incubated with 0.2 M 2-mercaptoethanol or 1 mM phenylmethylsulfonyl fluoride (PMSF) for 30 min at 37°C before the hemagglutination assay.

Hemagglutination assays.

Rabbit erythrocytes were used for the determination of hemagglutination activity during the isolation procedures and for inhibition assays. Fifty microliters of serial twofold dilutions of the fungal extracts was mixed with an equal volume of a 1% erythrocyte suspension in wells of U-shaped microtiter plates. After gentle shaking, the plates were allowed to settle at room temperature for 1 h and agglutination was recorded visually. PBS was used as a negative control. The hemagglutination titer was defined as the reciprocal of the highest dilution of the fungal extract that yielded visible hemagglutination activity. One hemagglutination unit (HAU) was defined as the amount of fungal extract which causes complete agglutination under the aforementioned conditions. The specific hemagglutination activity was recorded as the number of hemagglutination units per microgram of protein.

Hemagglutination inhibition assays.

Hemagglutination inhibition assays were performed by mixing 25 μl of an appropriate dilution of the fungal extract (corresponding to 4 HAU) in microtiter plates with an equal volume of serial twofold dilutions of potential inhibitors (see below). After 30 min of incubation at room temperature, 50 μl of a 1% erythrocyte suspension was added to each well and plates were allowed to settle at room temperature for 1 h. Controls contained 25 μl of PBS instead of the inhibitor solution. The MIC was defined as the lowest concentration of carbohydrate or glycoprotein required for complete inhibition of the hemagglutination.

The following sugars (stock solutions at 400 mM in PBS) and glycoproteins (stock solutions at 10 mg/ml in PBS), all from Sigma, were tested as inhibitors: d-glucose, d-galactose, d-mannose, d-fructose, d- and l-fucose, l-rhamnose, d-glucosamine, d-galactosamine, N-acetyl-d-glucosamine, N-acetyl-d-galactosamine, d-trehalose, d-lactose, d-melibiose, d-maltose, d-saccharose, N-acetyl-neuraminic acid (NANA), colominic acid, bovine thyroglobulin, human fibrinogen, bovine fetuin, bovine submaxillary mucin, bovine asialomucin, and human orosomucoid. Bovine mucin was also used after hydrolysis of its O-acetylated groups (35).

Purification procedures. (i) Fractionation by gel filtration.

Fungal extract I was applied to a Highload Superdex TM 75 gel filtration column (60 by 1.6 cm; Pharmacia) previously equilibrated with PBS. Elution was carried out at room temperature. Fractions of 4 ml were collected at a flow rate of 1 ml/min. They were monitored for protein content at 280 nm and for hemagglutination activity as previously described. The reactive fractions were pooled, and ammonium sulfate was added to 80% saturation. After 1 h of incubation at 4°C, the precipitate was collected by centrifugation, dissolved in 20 mM Tris-HCl buffer (pH 8.5), and dialyzed against the same buffer for 48 h.

(ii) Ion-exchange chromatography.

The lectin was then purified by ion-exchange chromatography on a Mono Q HR 5/5 column (Pharmacia) equilibrated with a 20 mM Tris-HCl buffer (pH 8.5). Elution was carried out with the same buffer at a flow rate of 1 ml/min by a stepwise increase in the concentration of NaCl (25 mM to 3 M). Fractions of 4 ml were collected and checked for hemagglutination activity.

(iii) Hydrophobic-interaction chromatography.

For hydrophobic-interaction chromatography, the active fractions were pooled and proteins were precipitated by slow addition of an equal volume of a 50 mM phosphate buffer containing 2.8 M ammonium sulfate. After 1 h of incubation at 4°C, insoluble residues were removed by centrifugation at 17,000 × g for 30 min. The supernatant was then loaded on a phenyl-Superose HR 5/5 column (Pharmacia) previously equilibrated with 50 mM phosphate buffer containing 1.4 M ammonium sulfate. Elution was carried out at a flow rate of 0.5 ml/min by a stepwise decrease of ammonium sulfate concentration until a 0.1 M concentration was achieved. The residual bound material was then eluted by using successively 150 mM phosphate buffer, 20% ethanol, and distilled water. Fractions of 4 ml were collected, dialyzed, and checked for hemagglutination activity.

Electrophoresis.

After each purification step, an aliquot of the fractions exhibiting the hemagglutination activity was precipitated with 10% trichloroacetic acid and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (12% acrylamide) according to the method of Laemmli (21). The gels were stained with Coomassie brilliant blue R 25. The purity of the lectin was confirmed by silver staining. The molecular masses of protein standards were determined with a low-molecular-mass electrophoresis calibration kit (Pharmacia).

RESULTS

Specific hemagglutination activity of the fungal extracts.

The presence of a lectin in the crude extracts prepared from resting or swollen conidia, germ tubes, and mycelium of strain CBS 113.26 was detected by hemagglutination assays with rabbit erythrocytes. The lectin activity was recovered in all extracts, whatever the extraction procedure used (Table 1). However, extracts from resting conidia exhibited the highest specific hemagglutination activity. Then a decrease in the hemagglutination activity was observed as the germination occurred, the lowest activity being observed in germ tubes and in 5-day-old mycelium.

TABLE 1.

Hemagglutination activity of the different morphological stages of A. fumigatus

Morphological stage	Fungal extract	Protein concn (μg/ml)	Hemagglutination activity (HAU/ml)	Sp act (HAU/μg)
Resting conidia	I	27.5	800	29.1
	II	9.6	800	83.4
Swollen conidia	I	26.6	400	15
	II	35.5	400	11.3
Germ tubes	I	284.4	400	1.4
	II	50.9	400	7.9
Mycelium	I	194.8	800	4.1
	II	666.6	850	1.3

The efficiency of the extraction of the hemagglutinin was dependent on the morphological stage. In resting conidia, the best results were obtained by sonication, whereas grinding in liquid nitrogen was more efficient for mycelium. However, preliminary tests showed that purification of the hemagglutinin required important amounts of fungal material. Therefore, a homogenate of frozen mycelium (fungal extract I) was used throughout for subsequent experiments.

Hemagglutination inhibition by various carbohydrates and glycoconjugates.

Various sugars and glycoconjugates were tested for their ability to inhibit the agglutination of rabbit erythrocytes by the lectin. Glucose, galactose, mannose, fructose, fucose, rhamnose, glucosamine, galactosamine, N-acetyl-d-glucosamine, N-acetyl-d-galactosamine, lactose, melibiose, trehalose, and saccharose, at the highest concentration used, did not inhibit the hemagglutination activity of fungal extract I from strain CBS 113.26. Conversely, it was inhibited by NANA and the sialic acid-rich glycoproteins, i.e., bovine submaxillary mucin, thyroglobulin, fibrinogen, and fetuin (Table 2). Moreover, similar results were obtained with the ALP-nonproducing strain Δ18.

TABLE 2.

Inhibition of hemagglutination by NANA and sialoglycoproteins

Inhibitor	MIC (equivalent NANA-bound concn)a
NANA	25 mM
Bovine mucin	83 μg/ml (32 μM)
Treated bovine mucin	166 μg/ml (66 μM)
Asialomucin	2.5 mg/ml (96 μM)
Bovine thyroglobulin	625 μg/ml (28 μM)
Human fibrinogen	5 mg/ml (86 μM)
Bovine fetuin	666 μg/ml (86 μM)
Human orosomucoid	—b
Colominic acid	—

^aEquivalent concentration of NANA bound evaluated on the basis of data from the literature (15, 38).

^b—, absence of inhibition at the higher concentration used (10 mg/ml).

The sialoglycoproteins tested were more potent inhibitors than NANA, indicating that the lectin presented a higher affinity for linked sialic acids. Among them, bovine submaxillary mucin, which presented both terminal 9-O-acetyl and 5-N-acetyl sialic acids, was the most potent inhibitor (Table 2). Base treatment specific for hydrolysis of the O-acetyl groups of sialic acids slightly affected the inhibitory potency of mucin, whereas its desialylation induced a strong reduction. The minimum concentration required for complete inhibition at 4 HAU was 83 μg/ml for bovine mucin, whereas it was 2.5 mg/ml for its desialylated form. This could be explained by the fact that desialylation was incomplete (about 90%, according to the manufacturer). Indeed, when calculated in NANA-bound equivalent concentrations, the results were similar for all active glycoproteins, ranging from 28 to 96 μM (Table 2).

Orosomucoid did not inhibit the lectin. Unlike the other sialoglycoproteins tested, it contains only N-glycosidically linked carbohydrate chains. Likewise, colominic acid, a homopolymer [poly-α-(2,8)-NANA], had no inhibitory effect at the concentration of 10 mg/ml, indicating that the lectin had less affinity towards subterminal sialic acid than terminal residues. Finally, treatment of the crude extract with 0.2 M 2-mercaptoethanol did not affect the hemagglutination activity.

Purification of the hemagglutinin.

The crude fungal extract was applied to a gel filtration column. Elution with PBS yielded four protein peaks. The hemagglutination activity eluted in the second peak, corresponding to fractions 35 to 45 (Fig. 1). The active fractions were pooled and analyzed by SDS-PAGE. After Coomassie blue staining, a large number of molecular species were visualized, ranging from 10 to 94 kDa (Fig. 2A, lane 2).

FIG. 1.

Elution protein profile (•) and hemagglutination activity (○) from gel filtration chromatography.

FIG. 2.

SDS-PAGE under denaturing conditions of the active fractions from the different steps of the purification procedure. Crude fungal extract (lane 1) and active fractions obtained from gel filtration (lane 2), ion-exchange (lanes 3), and hydrophobic-interaction (lanes 4) chromatographies are shown. The proteins were visualized by Coomassie blue staining (A) or by silver staining (B). The molecular masses (MM) of protein standards (in kilodaltons) are indicated on the left.

The lectin was further purified by ion-exchange chromatography on a Mono Q HR 5/5 column. Elution was carried out with increasing concentrations of NaCl. The hemagglutination activity was found in fractions corresponding to elution by 25, 50, and 75 mM NaCl. SDS-PAGE analysis of the pooled reactive fractions showed after Coomassie blue staining numerous polypeptide bands, with three major proteins of 32, 35, and 40 kDa (Fig. 2A, lane 3).

Finally purification was achieved by hydrophobic-interaction chromatography, by using for elution a stepwise decreasing concentration of ammonium sulfate in phosphate buffer. The lectin activity was recovered in the elution peak corresponding to 0.1 M ammonium sulfate. This fraction examined by SDS-PAGE showed a single band with an apparent molecular mass of 32 kDa both in the presence and in the absence of the reducing agent (Fig. 2A, lane 4). Silver staining of the gel loaded with the reactive fractions from the last two purification steps demonstrated the purity of the final product and the interest of the hydrophobic-interaction chromatography (Fig. 2B, lanes 3 and 4). During these purification steps, the specific activity of the lectin increased 21.7-fold, with an overall recovery of 0.96% (Table 3).

TABLE 3.

Summary of purification of A. fumigatus hemagglutinin

Purification step	Total vol (ml)	Protein concn (μg/ml)	Hemagglutinating activity (HAU/ml)	Recovery (%)	Sp act (HAU/μg)	Purification factor (fold)
Crude extract	6.5	10,000	5,120	100	0.5	1
Gel filtration	38	175	640	73	3.7	7.1
Ion-exchange chromatography	6.8	26	160	3.3	6.2	11.9
Hydrophobic-interaction chromatography	4	7	80	0.96	11.1	21.7

Relationship between the hemagglutination and serine protease activities.

The relationship between hemagglutination and serine protease activities was investigated by using PMSF, a potent inhibitor of all serine proteases, and a serine protease-negative mutant (ALP-nonproducing Δ18). Preincubation of fungal extract I in 1 mM PMSF did not affect the hemagglutination. Moreover, no difference in hemagglutination activity was found between ALP-producing (CBS 113.26) and ALP-nonproducing (Δ18) strains.

DISCUSSION

Lectins responsible for adherence and cellular colonization have been widely demonstrated among living organisms. Recognition of glycoconjugates is an important event in biological systems and is frequently mediated by carbohydrate-protein interactions. However, very few data are available on hemagglutinins produced by the aspergilli (Ishimaru et al., letter). Here we demonstrated that A. fumigatus possesses a 32-kDa lectin that agglutinates rabbit erythrocytes.

First we searched for a hemagglutination activity in different fungal extracts and determined which saccharides or glycoproteins were able to inhibit hemagglutination. Fungal extracts obtained from resting conidia presented the highest specific hemagglutination activities, particularly that obtained by sonication, a treatment known to alter the superficial layers of the fungal cell wall (8). These results led to the conclusion that the lectin was preferentially concentrated in the cell wall of conidia, where it could provide a mechanism for their attachment to epithelial cells or to the extracellular matrix. In contrast, grinding in liquid nitrogen resulted in a greater specific hemagglutination activity for mycelium, suggesting a preferential localization of the protein in the cytoplasm rather than in the cell wall. Nevertheless, production of specific antibodies directed against the lectin is needed to specify its localization in the different morphological stages of the fungus.

Inhibition hemagglutination assays performed with various carbohydrates or glycoproteins demonstrated the specificity of the lectin for sialic acid residues. Among the compounds tested, only NANA and the sialoglycoproteins inhibited the lectin activity. Sialic acids, which are often found as terminal sugars of glycoconjugates, play an important role in many biological mechanisms (17, 33, 34, 43, 44). In a previous report, it was demonstrated that NANA and sialyl lactose inhibited the binding of laminin and fibrinogen to A. fumigatus conidia (3), suggesting an interaction mediated by a sialic acid-specific lectin of the conidial wall. Interestingly, such a mode of recognition of extracellular matrix components has also been reported for various bacteria, for example Escherichia coli (45), Helicobacter pylori (13, 42), and Pseudomonas aeruginosa (32, 33). Among fungi, it has been demonstrated that Penicillium marneffei (12) and H. capsulatum (28) also possess a lectin specific for sialic acid. In contrast, for A. fumigatus a 32-kDa fucose-specific lectin has been identified recently (Ishimaru et al., letter). This lectin could provide a mechanism not only for the attachment of conidia to fucosylated antigens of the epithelial cell membrane but also for the recognition of matrix proteins through the fucose residues of their carbohydrate chains. Curiously, in our study, we did not observe any inhibition with fucose for the two strains that we used. This discrepancy may be related to differences in strains or culture conditions.

The main protein present in the eluted fraction from hydrophobic chromatography was characterized by SDS-PAGE as a 32-kDa polypeptide band. Since an extracellular ALP of 32 to 33 kDa belonging to the subtilisin subset of serine proteases has been isolated from different strains of A. fumigatus (23, 29), we examined if the hemagglutination activity is supported by ALP. Indeed, some bacterial or parasitic proteins have been reported to harbor both enzymatic and adhesive or lectin properties (1, 14, 24, 25). In this study, the absence of inhibition of the hemagglutination after pretreatment of the fungal extracts in the presence of PMSF indicated that both hemagglutination and protease activities were independent. In addition, an A. fumigatus mutant in which the gene encoding the serine protease was disrupted (ALP-nonproducing strain Δ18) exhibited a similar sialic acid-specific lectin activity, indicating that the two activities were supported by different proteins.

Together, these results suggest the presence at the conidial surface of a lectin specific for sialic acid residues. Given the potential role of laminin and fibrinogen in the adherence of A. fumigatus conidia and their high carbohydrate content with terminal sialic acid residues (19, 38), we are now investigating if the 32-kDa lectin is involved in the binding of these proteins.