Glycobiology Advance Access originally published online on January 12, 2007
Glycobiology 2007 17(4):401-410; doi:10.1093/glycob/cwl085
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Structure and role of sialic acids on the surface of Aspergillus fumigatus conidiospores
2 Department of Biological Sciences, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, V5A 1S6, Canada
3 Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, V5A 1S6, Canada
1 To whom correspondence should be addressed; Tel: +1-604-291-3441; Fax: +1-604-291-3496; e-mail: mmoore{at}sfu.ca
Received on October 22, 2006; revised on December 21, 2006; accepted on January 2, 2007
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Abstract |
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 Top Abstract IntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Aspergillus fumigatus is an opportunistic fungal pathogen that causes a life-threatening invasive fungal disease (invasive aspergillosis, IA) in immunocompromised individuals. The first step of pathogenesis is thought to be the attachment of conidia to proteins in lung tissue. Previous studies in our laboratory have shown that conidia adhere to basal lamina proteins via negatively charged sugars on their surface, presumably sialic acids. Sialic acids are a family of more than 50 substituted derivatives of a nine-carbon monosaccharide, neuraminic acid. The purpose of this study was 2-fold: (1) to determine the structure of sialic acids and the glycan acceptor on A. fumigatus oligosaccharides and (2) to determine the effect on the removal of sialic acids from conidia on conidial binding to the extracellular matrix protein fibronectin and phagocytosis of conidia by cultured macrophages and type 2 pneumocytes. Surface sialic acids were removed using Micromonospora viridifaciens sialidase or using acetic acid, mild acid hydrolysis. Lectin binding studies revealed that the majority of conidial sialic acids are
2,6-linked to a galactose residue. High-pressure liquid chromatography of derivatized sialic acids released from conidia revealed that unsubstituted N-acetylneuraminic acid is the predominant sialic acid on the surface of conidia. Enzymatic removal of sialic acid significantly decreased the binding of conidia to fibronectin by greater than 65% when compared with sham-treated controls. In addition, removal of sialic acids decreased conidial uptake by cultured murine macrophages and Type 2 pneumocytes by 33% and 53%, respectively. Hence, sialylated molecules on A. fumigatus conidia are ligands for both professional and nonprofessional phagocytes. Key words: adhesion / fibronectin / high-pressure liquid chromatography / phagocytosis / virulence factor
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Introduction |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Aspergillus species are saprophytic fungi that are found in soil, water, and decaying organic matter. There are more than 200 species of Aspergillus; however, only Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, and Aspergillus terreus are associated with invasive disease in humans (Latgé 1999
). Interestingly, A. fumigatus is responsible for over 90% of Aspergillus infections (Denning 1998). It can cause a life-threatening invasive pulmonary aspergillosis, called invasive aspergillosis, IA, in immunocompromised individuals, such as bone marrow and solid organ transplant patients (Trullas et al. 2005), patients with hematological malignancies (Herbrecht et al. 2005), human immunodeficiency virus/acquired immune deficiency syndrome patients (Ruhnke 2004), and patients with chronic granulomatous disease (Rosenzweig and Holland 2004). IA is now a leading cause of death in medical centers worldwide (Marr et al., 2004). Antifungal drugs are used to treat IA; however, the current success rate of treatment averages only 53% (Herbrecht et al. 2002) with mortality ranging between 54% and 84% in haematopoetic stem cell recipients and between 20% and 67% in solid organ transplant recipients (Morgan et al., 2005). Because of the severity of Aspergillus infection and the lack of satisfactory antifungal therapy, it is critical to better understand its mechanisms of pathogenesis.
Sialic acids are a family of more than 50 substituted derivatives of a nine-carbon monosaccharide, neuraminic acid (Angata and Varki 2002; Schauer 2004). Diversity in sialic acid structure is generated through substitutions at the hydroxyl and amino groups located on carbons 4, 5, 7, 8, and 9 (Angata and Varki 2002; Schauer 2004). Sialic acids usually occur -ketosidically linked to glycoproteins and glycolipids and are often positioned as terminal monosaccharides in these glycoconjugates (Schauer 2004). Sialic acids have been found to play important roles in microbial pathogenesis. For example, binding of influenza A and B viruses to host cells is accomplished by the specific recognition of the host sialic acid linkage by the virus (Suzuki 2005). In addition to recognizing the sialic acid linkage, viruses belonging to the family Orthomyxoviridae and Coronaviridae are able to recognize and bind acetylated sialic acids (Strasser et al. 2004). Several bacterial species incorporate sialic acid into their glycoconjugates, and surface sialylation has been found to aid in the evasion of the bacterium from the host immune response by inhibiting the activation of the alternate complement pathway (Ram et al. 1998). Several human fungal pathogens have also been found to express sialic acids on their cell surface. These include Fonsecaea pedrosoi (Souza et al. 1986), Cryptococcus neoformans (Rodrigues et al. 1997), Paracoccidioides brasiliensis (Soares et al. 1993, 1998), Candida albicans (Soares et al. 2000), Sporothrix schenckii (Alviano et al. 1982), Pneumocystis carinii (De Stefano et al. 1990), and Aspergillus fumigatus (Wasylnka et al. 2001). N-acetylneuraminic acid (Neu5Ac) was the most common sialic acid derivative encountered; however, 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac2) has been found on the surface of C. neoformans and F. pedrosoi (Alviano et al. 2004; Rodrigues et al. 1997).
Previous work in our laboratory has identified negatively charged carbohydrates on the surface of A. fumigatus conidia (Wasylnka and Moore 2000). Subsequent analysis revealed that those sugars were sialic acids (Wasylnka et al. 2001); however, the experimental conditions used for isolation would not have preserved the structure of most substituted sialic acids. We have also shown that pathogenic Aspergillus species had greater amounts of sialic acids on their surface than nonpathogenic Aspergillus species; however, the role of these sialic acids in microbial pathogenesis has not been established (Wasylnka et al. 2001). The initial step of infection is thought to be the adhesion of A. fumigatus conidia to host lung cells or to components of the lung extracellular matrix (ECM) (Bouchara et al. 1994). Pathogenic Aspergillus species also bound to the ECM protein, fibronectin, to a greater extent than nonpathogenic Aspergillus species; therefore, we hypothesized that adhesion of A. fumigatus to fibronectin is mediated by the surface sialic acids (Wasylnka et al. 2001).
In addition to a potential role in adhesion, sialic acids have also been implicated in protecting C. neoformans and S. schenckii from the host immune response: removal of surface sialic acids using sialidase increased the level of phagocytosis 2- and 8-fold, respectively (Oda et al. 1983; Rodrigues et al. 1997). Furthermore, removal of surface sialic acids from F. pedrosoi increased the level of association between the fungus and neutrophils (Alviano et al., 2004). We have previously shown that A. fumigatus conidia are efficiently internalized by cultured human type II pneumocytes and murine macrophages (Wasylnka and Moore 2002), but the importance of sialic acids in the phagocytosis of A. fumigatus conidia is not known.
The specific aims of the current research were: (1) to characterize the linkage and the structure of terminal sialic acids on the surface of A. fumigatus conidia as well as to identify the sub-terminal monosaccharide, (2) to determine whether sialic acids on the surface of A. fumigatus conidia mediate the binding between conidia and the ECM protein, fibronectin, and (3) to assess whether the removal of sialic acids from conidia affected the extent of internalization by cultured murine macrophages and type II pneumocytes.
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Results |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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Surface sialic acids in A. fumigatus are predominantly
2,6-linked to the sub-terminal carbohydrateTo identify the linkage of conidial surface sialic acids to the sub-terminal carbohydrate, a biotin-labeled lectin-binding assay using flow cytometry was performed. Sambucus nigra agglutinin (SNA), which reacts with
2,6-linked sialic acids (Shibuya et al. 1987), and Maackia amurensis agglutinin (MAA), which reacts with 2,3-linked sialic acids (Knibbs et al. 1991), were incubated with conidia, labeled with a secondary fluorophore, Streptavidin-Oregon green, and analyzed by flow cytometry. A range of lectin concentrations were used (0–800 µg/mL) as well as a negative control consisting of Candida tropicalis cells incubated with MAA. C. tropicalis was previously shown to be unreactive with MAA (Munoz et al. 2003). SNA bound to A. fumigatus conidia, whereas MAA bound to a small extent, but only at a very high lectin concentration (800 µg/mL). This indicated that the surface sialic acid is almost exclusively linked to the underlying carbohydrate by an 2,6 linkage (Figure 1).
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To determine the specificity of SNA binding, the lectin-binding assay was performed as described above with the addition of 38 mM
2,6-sialyllactose (Sigma) to compete with conidial sialic acids for SNA. The results indicated that 2,6 sialyllactose efficiently competed with conidial sialic acids for SNA, confirming the specificity of SNA binding to conidia (data not shown).
Previous attempts to remove sialic acids from A. fumigatus conidia using commercially available Clostridium perfringens and Arthrobacter ureafaciens sialidases resulted in only half of the sialic acids being removed (Wasylnka et al. 2001). Micromonospora viridifaciens sialidase has been shown to effectively cleave sialic acids in 2,3, 2,6, and 2,8 linkages (Sakurada et al. 1992; Watson et al. 2003). Therefore, conidia were treated with M. viridifaciens sialidase or incubated under the same conditions without the addition of enzyme, and the extent of sialic acid release was assessed by lectin binding with SNA monitored by flow cytometry and fluorescence microscopy. These analyses showed that approximately 90% of surface sialic acids were removed by M. viridifaciens sialidase (Figure 2). This provided us with a useful tool to test the effect of sialic acid removal on adhesion and uptake. Moreover, the results confirmed the specificity of SNA binding since enzymatic removal of sialic acids eliminated the SNA binding (Figure 2).
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The sub-terminal carbohydrate on A. fumigatus conidia is galactose
To determine the identity of the underlying monosaccharide, M. viridifaciens sialidase-treated A. fumigatus conidia were incubated with PNA, a lectin specific for exposed galactose residues (Lotan et al. 1975
). Arachis hypogaea agglutinin (PNA)-reactive material was observed on conidia only after sialidase treatment revealing that the sub-terminal monosaccharide in A. fumigatus is galactose (Figure 3). Figure 3 also shows that the removal of the negatively charged sialic acids caused the conidiospores to agglutinate.
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High-pressure liquid chromatography (HPLC) and matrix assisted laser desorption ionization (MALDI) mass spectroscopy reveal that Neu5Ac is the major sialic acid on the surface of A. fumigatus conidia
To determine whether substituted sialic acids exist on the surface of A. fumigatus conidia, a mild acid treatment was used to release the terminal sialic acids from conidia. This treatment releases terminal sialic acids from glycoconjugates without destroying the substitutions that may be present on the sialic acid molecule (Varki and Diaz 1984
). Released sialic acids were then derivatized with 1,2-diamino-4,5-methylenedioxybenzene (DMB), dihydrochloride and separated by HPLC, and retention times are compared with two standards: an acid hydrolysate of bovine submaxillary mucin (BSM), which is a rich source of Neu5Ac and O-acetylated sialic acids, with the exception of the 4-O-acetylated derivative (Reuter et al. 1983) and an acid hydrolysate of horse serum, which is a source of 4-O-acetylated sialic acid (Manzi et al. 1990). HPLC revealed three DMB-reactive compounds in the mild acid hydrolysate of A. fumigatus conidia; however, only one of these peaks eluted with a relative retention time that corresponded with peaks in the standards (Figure 4A). Fractions corresponding to each of the three peaks were collected, dried, and pooled for mass spectroscopic analysis. MALDI mass spectroscopy revealed that peak 1 was Neu5Ac by the presence of molecular ions at m/z = 426 [M + H]+, m/z = 448 [M + Na]+, and m/z = 464 [M + K]+, the resulting masses of DMB-derivatized Neu5Ac and sodium and potassium adducts, respectively (Figure 4B). Peaks 2 and 3 did not elute with relative retention times corresponding with those of the standards, and MALDI mass spectroscopic data of those peaks was inconsistent with that of any known sialic acid derivative: mass spectral data revealed that peaks 2 and 3 were of lower mass than Neu5Ac (peak 2, m/z = 394 [M + H]+; peak 3, m/z = 387 [M + H]+, data not shown). 2-Keto-3-deoxy-nonulosonic acid (KDN) is a structurally related -keto acid that is also found in glycoconjugates. However, under the conditions used in this study, KDN would elute before Neu5Ac, and KDN has a DMB derivatized mass of m/z = 385 [M + H]+. In addition, alkaline hydrolysis neither eliminated peak 3 nor increased the size of the Neu5Ac peak, indicating that peak 3 is not likely to be an O-acetylated sialic acid derivative (data not shown). Although peak 2 was alkaline labile, there was no corresponding increase in the size of the Neu5Ac peak, further indicating that this peak was unlikely to be an O-acetylated sialic acid. Sialidase-released material was also derivatized and analyzed by HPLC and the results were identical to those of the acid-released material (data not shown). Hence, Neu5Ac was established as the predominant sialic acid on the surface of A. fumigatus conidia.
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Binding of A. fumigatus conidia to fibronectin-coated wells is decreased upon sialidase treatment of conidia
To evaluate whether sialic acids on A. fumigatus conidia are important in mediating their binding to the ECM protein, fibronectin, sialidase-treated or untreated conidia were incubated in fibronectin-coated wells, and the number of adherent conidia was assessed by light microscopy. Sialidase treatment of conidia reduced the level of binding of conidia to fibronectin-coated wells by greater than 65% compared with untreated spores (Figure 5). This confirms our hypothesis that sialic acids on the conidial surface are the major fungal ligand involved in adhesion to fibronectin.
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Phagocytosis of A. fumigatus conidia by cultured mouse macrophages and Type II pneumocytes is decreased upon sialidase treatment of conidia
To assess whether sialic acids are important to the uptake of conidia by cultured cells, sialidase-treated or untreated green fluorescent protein (GFP)-expressing A. fumigatus conidia were incubated with either a macrophage cell line (J774) or a human lung epithelial cell line (A549), and phagocytosis was allowed to proceed. The extent of conidial uptake for each condition was determined by subtracting the number of extracellular conidia (labeled both green and red) from the total conidia (labeled green only). As expected, the uptake of bound conidia by J774 cells was very efficient (74% uptake) and lower in the A549 cell line, which are not professional phagocytes (50% uptake). Sialidase treatment of conidia decreased the uptake by both cell lines: uptake by J774 cells and A549 cells was decreased by 33% and 53%, respectively (Figure 6A and B). Thus, sialylated molecules on A. fumigatus conidia are recognized by the receptors on both epithelial cells and macrophages. Interestingly, although the uptake was significantly decreased by sialidase treatment, in A549 cells, the binding of conidia did not decrease when sialic acids were removed. In fact, the number of adherent conidia significantly increased when they were treated with sialidase (Figure 6C). There was no significant difference in the level of conidial binding to J774 cells after sialidase treatment (Figure 6C). Therefore, no correlation was found between the extent of binding of conidia and their internalization by cultured pneumocytes and macrophages. These data suggest that, for A549 cells, sialidase treatment exposed additional adhesins on conidia that were able to bind to the host cells, but could not initiate internalization.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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This study has demonstrated that the majority of A. fumigatus sialic acids are unsubstituted sialic acids linked to an underlying galactose via an
2,6-linkage, with only trace amount of 2,3-linkage. The conidial sialic acids were shown to mediate the majority of binding between fibronectin and conidia. Finally, the removal of sialic acids from conidia reduced the uptake of conidia by J774 and A549 cells, suggesting that sialylated ligands are important in the recognition and phagocytosis of this pathogen.
HPLC of DMB-derivatized sialic acids showed three peaks, but only peak 1 was consistent with a known sialic acid, Neu5Ac. Studies have shown that DMB does not react with glucose, galactose, fructose, glucosamine, galactosamine, maltose, cellobiose, gentiobiose, lactose, fucose, ribose, deoxyribose, and acidic sugars such as uronic acids (Hara et al. 1989). DMB reacts with dicarbonyl compounds that are abundant and play important roles in metabolism (Hara et al. 1989) and peak 2 may represent such a compound. Several published studies also report an unidentified reagent peak with a relative retention time between that of Neu5,9Ac2 and Neu5,7(8),9Ac3 (Hara et al. 1989; Chatterjee et al. 2003; Lewis et al. 2006), corresponding to peak 3 in our study. It is likely that peaks 2 and 3 are artifacts of the conditions employed in this study and, therefore, we conclude that Neu5Ac is the major sialic acid on the surface of A. fumigatus conidia.
Basal lamina of diseased lungs have increased levels of deposited fibronectin (Torikata et al. 1985), which are thought to aid in repair of damaged epithelia by facilitating the attachment of cells required for re-epithelialization (Clark et al. 1982). Previous studies from our laboratory have shown that A. fumigatus conidia specifically bound to the positively-charged glycosaminoglycan (GAG)-binding domain of fibronectin and that the binding was inhibited by negatively-charged sugars (Wasylnka and Moore, 2000). The results of the present study confirm the hypothesis that sialic acids play a major role in conidial adhesion to ECM proteins. Given that there are increased levels of fibronectin in lung tissue upon injury (Torikata et al. 1985), and that lung injury is a known risk factor associated with IA (Bodey and Vartivarian 1989), surface sialic acids may mediate the initial adhesion step of infection. However, more work needs to be performed to establish whether this occurs in vivo.
It has been proposed that fungal sialic acids mask foreign epitopes and consequently protect fungal cells from phagocytosis. The removal of cell surface sialic acids from S. schenckii and C. neoformans by sialidase treatment resulted in an increased rate of phagocytosis by mouse peritoneal macrophages (Oda et al. 1983; Rodrigues et al. 1997). Additionally, sialidase treatment of F. pedrosoi conidia resulted in a greater number of interactions between conidia and neutrophils (Alviano et al. 2004).
Professional phagocytes have receptors for galactose or N-acetylgalactose residues, which are usually the sub-terminal monosaccharides to which sialic acids are linked (Linehan et al. 2000). An increase in the uptake was attributed to the presence of galactose receptors on the phagocyte surface that are able to recognize the exposed galactose residues and hence internalize the organism more efficiently (Oda et al. 1983; Rodrigues et al. 1997; Alviano et al. 2004). For C. neoformans and F. pedrosoi, it was proposed that sialic acids protect the organism during the early stages of infection until the organism can reach a more mature and highly resistant stage. In the present study, sialidase treatment of A. fumigatus conidia yielded the opposite result; uptake by both the J774 macrophage cell line and the A549 lung epithelial cell line decreased (by 33% and 53%, respectively). Therefore, in A. fumigatus, sialic acids may not play a comparable protective role in the same way as they do in other fungal organisms. Rather, binding of phagocytes to sialylated ligands on the conidial surface enhanced the uptake. In addition to galactose receptors, macrophages have many other receptors on their cell surface that mediate cell–cell interactions (Linehan et al. 2000).
Sialoadhesin (siglec 1) is one among them (Linehan et al. 2000). Sialoadhesin is part of a family of sialic acid-binding immunoglobulin-like lectins (siglecs) and is found exclusively on macrophages (Angata and Brinkman-Van der Linden 2002). Sialoadhesin specifically recognizes 2,3- and 2,6-linked Neu5Ac and to a lesser extent, 2,8-linked Neu5Ac (Angata and Brinkman-Van der Linden 2002). The molecular basis for sialic acid recognition has been elucidated by X-ray crystallography and nuclear magnetic resonance spectroscopy. It was found that sialoadhesin forms important hydrogen bonds with the glycerol side chain of Neu5Ac, specifically, with the hydroxyl groups on carbons 7, 8 and 9 (May et al. 1998). Consequently, sialoadhesin does not recognize 9-O-acetylated sialic acids (Kelm et al. 1994, 1998; Shi et al. 1996). Additionally, sialoadhesin does not recognize Neu5Gc, a common sialic acid derivative that differs from Neu5Ac in that the N-acetyl group located on carbon 5 is hydroxylated (Kelm et al. 1994, 1998). S. schenckii was found to have Neu5Gc (Oda et al. 1983) and both C. neoformans and F. pedrosoi possess 9-O-acetylated sialic acids (Rodrigues et al. 1997; Alviano et al. 2004). Therefore, in contrast to A. fumigatus, the modified sialic acids on these fungal pathogens would not be recognized by sialoadhesin and consequently, phagocytosis was inhibited. In A. fumigatus, sialidase treatment of conidia exposed galactose residues that are potentially recognized by macrophages via their galactose receptors (Linehan et al. 2000); however, it appears that sialic acid recognition and uptake occurred to a greater extent.
Sialidase treatment also decreased the uptake of conidia by the human type II pneumocyte cell line (A549). Previous findings in our laboratory showed that A. fumigatus conidia are taken up by A549 cells into early endosomes and ultimately form a phagolysosome. A portion of the internalized conidia survived and germinated within the A549 phagolysosome (Wasylnka and Moore 2003). Since the removal of conidial sialic acids significantly decreased the number of conidia internalized by A549 cells, sialylated fungal molecules are important in mediating the uptake by these cells. Such internalization may represent a strategy to evade the host immune response. The receptor on the epithelial cell is presently unknown. Unlike sialoadhesin, which is present in macrophages, sialic acid-binding lectins have not yet been reported in epithelial cells.
In conclusion, unlike other pathogenic fungi described to date, Neu5Ac is the predominant sialic acid on the surface of A. fumigatus conidia; no evidence for 9-O-acetylated derivatives was obtained. Neu5Ac is linked to an underlying galactose residue via an 2,6 linkage. Sialic acids on the conidial surface mediate the binding to the ECM protein, fibronectin, likely via interactions with the glycosaminoglycan binding domain on fibronectin. Finally, sialylated molecules on A. fumigatus conidia are recognized by cultured macrophages and lung epithelial cell lines and enhance the uptake of conidia into the endosomal system of the host cell.
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Materials and methods |
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Fungal strains and growth conditions
Two strains of A. fumigatus were used in this study: A. fumigatus American Type Culture Collection (ATCC, Manassas, VA) 13073 and the GFP-expressing strain of A. fumigatus ATCC 13073 previously constructed in our lab (Wasylnka and Moore 2002
). Candida tropicalis (CBS Fungal Biodiversity Center #94, Utrecht, The Netherlands) was used as a negative control for MAA binding during the linkage analysis. All strains were grown on MYPD agar (0.3% yeast extract, 0.3% malt extract, 0.5% peptone and 0.5% dextrose) for 3 days at 37 °C or 5 days at 28 °C until cells were fully mature. Conidia or cells were harvested by adding phosphate-buffered saline (PBS) (pH 7.4) containing 0.05% Tween 20 (PBS-T) directly onto the plate and scraping with a sterile cotton swab. The conidial suspension was then vortexed vigorously to break apart chains of conidia and filtered through sterile glass wool to remove hyphae. Conidia or cells were then washed 3 times with PBS, resuspended in PBS, and counted with a haemocytometer.
Sialidase treatment of A. fumigatus conidia to release surface sialic acids
A. fumigatus conidia (1 x 108) were washed once with 16 mM sodium tartrate buffer, pH 5.2, and resuspended in 25 µL of 16 mM sodium tartrate buffer supplemented with 0.1% bovine serum albumin (BSA) (Sigma, Oakville, ON, Canada). Recombinant Micromonospora viridifaciens sialidase (74 µg in 100 µL of 16 mM sodium tartrate buffer) was added to conidia and allowed to incubate between 2 and 3 h at 50 °C (Watson et al. 2003). After sialidase treatment, conidia were washed 3 times with PBS and stored at 4 °C for further analysis. The supernatant was clarified by centrifugation and filtered through a 10 000 molecular weight cut-off filter (Millipore, Billerica, MA) and stored at –20 °C.
Mild acid hydrolysis of A. fumigatus conidia to release surface sialic acids
A. fumigatus conidia (10 x 1010) were washed twice with distilled deionized H2O, and 1 x 1010 conidia were add to 50-mL plastic tubes. To each tube, 35 mL of 2.5 M acetic acid was added and the suspension was incubated for 5.5 h at 90 °C with shaking on every half hour. After incubation, tubes were immediately cooled with ice, clarified by centrifugation and lyophilized. Dried hydrolysates were resuspended in 500 µL H2O, filtered through a 10 000 mol. w. cut-off filter and stored at –20 °C.
Lectin binding assays
PNA, SNA, and MAA (all from Sigma) (1 mg in 1 mL PBS) were biotinylated by incubating with 300 µg of N-hydroxysuccinimidobiotin, long chain (Pierce Biotechnology, Inc., Rockford, IL) in dimethylformamide for 1 h at room temperature. Biotinylated lectins were then dialyzed against 4 mL PBS in 5000 mol. w. cut-off filters (Amicon) and resuspended in 500 µL PBS for a final stock concentration of 2000 µg/mL for each lectin.
To assess the extent of removal of surface sialic acids by sialidase treatment or mild acid treatment, lectin binding was used. Sialidase-treated or mild acid treated A. fumigatus conidia (1 x 107) were blocked with 45 µL PBS containing 10% goat serum for 1 h at room temperature. After 1 h, 5 µL of SNA stock solution (2000 µg/mL) was added for a final lectin concentration of 200 µg/mL and allowed to incubate for 1 h at room temperature. Conidia were then washed 3 times with PBS to remove unbound lectin, and bound lectin was detected by incubation with 25 µL of 1% Streptavidin-Oregon green (Molecular Probes, Burlington, ON, Canada) in PBS with 10% goat serum for 1 h at room temperature in the dark. After incubation, samples were washed 3 times with PBS, resuspended in an appropriate amount of PBS and mounted onto glass slides. Samples were viewed with an Olympus VANOX AHBS3 microscope (Olympus America, Inc., Center Valley, PA) equipped with epifluorescence at 1000x magnification. Bright field and fluorescence images were captured with a Sony 950 camera (Sony Canada Ltd, Toronto, ON, Canada) using Northern Eclipse imaging software (Empix Imaging Inc., Mississauga, ON, Canada). The extent of sialic acid removal was also assessed by flow cytometry. Flow cytometry was performed on a Coulter EPICS Elite Esp flow cytometer (Beckman-Coulter Inc., Mississauga, ON, Canada) using a 488-nm laser for excitation energy and a 550-nm dichroic filter capturing an emission band at 525 nm. A total of 10 000 events were captured for each sample.
To determine the linkage of surface sialic acids to the underlying carbohydrate, A. fumigatus conidia or C. tropicalis cells (1 x 107) were incubated with SNA or MAA (C. tropicalis cells were incubated with MAA only), labeled with Streptavidin-Oregon green, and analyzed by flow cytometry, as described in the Results section.
For competition experiments, conidia were incubated with SNA, as described in the Results section with the exception that 38 mM 2,6-sialyllactose was added along with SNA to compete with conidial sialic acids for SNA.
To determine the identity of the sub-terminal carbohydrate, sialidase-treated or untreated A. fumigatus conidia were incubated with PNA and visualized with Streptavidin-Oregon green using fluorescence microscopy as described in the Results section.
Identification of surface sialic acids from A. fumigatus by fluorometric HPLC and MALDI mass spectroscopy
Mild acid or enzyme hydrolysates of A. fumigatus were derivatized with DMB (Dojindo Molecular Technologies, Inc., Gaitherslow, MD) by resuspending the dried hydrolysates in 100 µL H2O and adding 25 µL of this solution to 100 µL of DMB solution (7 mM DMB, 0.75 M ß-mercaptoethanol, and 18 mM sodium hydrosulfite in 1.4 M acetic acid). This solution was heated for 2.5 h at 56 °C in the dark and stopped by cooling on ice.
DMB-derivatized samples were directly separated using a Waters 600E HPLC system (Waters Corporation, Mississauga, ON, Canada) with a reverse phase C-18 column (250 x 4.6 mm, 5 µm particle size, Phenomenex, Morrence, CA) using isocratic elution with a solvent system of H2O–acetonitrile–methanol (84/9/7, v/v/v) at a flow rate of 0.9 mL/min. DMB-derivatized sialic acids were detected with a Hewlett Packard 1046A programmable fluorescence detector (Hewlett-Packard Development Company, L.P., Palo Alto, CA) using an excitation wavelength of 246 nm and emission wavelength of 442 nm. Authentic N-acetylneuraminic acid (Sigma) and mild acid hydrolysates of BSM (Sigma), and horse serum (Sigma), which are rich sources of modified sialic acids, were used as standards.
For MALDI mass spectroscopy, fractions corresponding to peaks of interest were collected into plastic tubes, dried, resuspended in a minimal volume of H2O, and pooled. Samples were mixed with an equal volume of 2,5-dihydroxybenzoic acid matrix solution (10 mg/mL in H2O–acetonitrile, 50/50, v/v) (Sigma). Mass spectra were collected with a PerSeptive Biosystems MALDI mass spectrometer (Applied Biosystems, Streetsville, ON, Canada) in the positive ion mode using an average of 100 laser shots, an accelerating voltage of 20 000 V, mass range of between 0 and 4000 Da and a delay time of 150 ns.
Alkaline hydrolysis of O-acetylated sialic acids
To confirm sialic acid O-acetylation, sialic acids were de-O-acetylated by treating the sialic acid-containing samples with 0.2 M NaOH for 30 min on ice (Varki and Diaz 1984). For the 4-O-acetylated sialic acid derivative, these conditions are not sufficient for de-O-acetylation, therefore, for this derivative, 0.2 M NaOH was added to the samples and the samples were incubated for 45 min at 37 °C (Tiralongo et al. 2000). O-acetylated sialic acids were confirmed based on their relative retention times as well as by an observed decrease in the size of the peak corresponding to the O-acetylated sialic acid, and a corresponding increase in the size of the parent sialic acid peak (either Neu5Ac or Neu5Gc) upon alkaline treatment.
Fibronectin adhesion assay
Fibronectin adherence assays were performed in eight-chamber glass slides (Becton Dickinson, Oakville, ON, Canada) by coating the slides with 300 µL of 50 µg/mL fibronectin (Sigma) in PBS and incubating at 37 °C for 1 h and then at 4 °C overnight. The following day, slides were blocked with 500 µL/well PBS with 0.1% BSA (Sigma) for 1 h at 37 °C. After 1 h, 1 x 107 sialidase-treated or untreated A. fumigatus 13073 conidia were added to the wells and incubated at 37 °C for 1 h. Nonadherent cells were removed by washing the wells 3 times with 200 µL PBS-T. After washing, cells were fixed with 300 µL 2.5% glutaraldehyde in PBS at room temperature for 1 h. Cells were visualized by light microscopy using an Olympus VANOX AHBS3 microscope at 1000x magnification. Bright field images were captured with a Sony 950 camera using Northern Eclipse imaging software. Five random fields for each well were counted. Each experiment was performed 3 times independently, and the results reported are the mean ± standard deviation of the three experiments. The Student's t-test was used assess the difference in binding of sialidase-treated and untreated conidia to fibronectin-coated wells.
Phagocytosis assays using A549 lung epithelial and J774 macrophage cell lines
Mouse macrophage (J774) and type II pneumocyte (A549) cell lines were obtained from ATCC and maintained on RPMI 1640 medium containing 10% fetal calf serum (FCS) (v/v) (Canadian Life Technologies, Burlington, ON, Canada) at 37 °C in a humidified 5% CO2 incubator. A549 cells were seeded at 2.5 x 105 cells/well in 1 mL of RPMI 1640 media supplemented with 10% FCS on 12-mm number 1 coverslips in 24-well plates (Fisher Scientific, Ottawa, ON, Canada) and grown for 16 h at 37 °C. J774 cells were seeded at 2.5 x 105 cells/well for 2 h at 37 °C in the same media. After growth, cells were blocked with 1 mL RPMI 1640 containing 10% FCS and 0.5% BSA at 37 °C for 1 h. After blocking, cells were infected with 2 x 106 GFP-expressing A. fumigatus 13073 conidia either treated or untreated with sialidase (as described in Sialidase treatment of A. fumigatus conidia) in 1 mL RPMI 1640 with 10% FCS and allowed to incubate at 37 °C for 1 or 3 h for J774 cells or A549 cells, respectively. After incubation, plates were put on ice to stop phagocytosis, and nonadherent conidia were removed by washing the wells 3 times with 1 mL PBS-T. Extracellular spores were labeled using a rabbit anti-Aspergillus primary antibody raised against A. fumigatus cell wall components, which was previously developed in our lab (Wasylnka and Moore 2002). Primary antibody was diluted 1 : 75 in PBS–10% goat serum, and 1 mL of this solution was added to each well and allowed to incubate on ice for 1 h. After 1 h, wells were washed 3 times with 1 mL PBS and cells were incubated with 1 mL goat anti-rabbit secondary antibody conjugated with Alexa Fluor 594 (Molecular Probes) diluted 1 : 575 in PBS–10% goat serum on ice for 45 min. Cells were then washed 3 times with 1 mL PBS and fixed with 500 µL PBS/4% paraformaldehyde on ice for 1 h. Coverslips were washed 3 times with 1 mL PBS, mounted onto slides with ProLong Antifade mounting media (Molecular Probes), sealed and stored at 5 °C until analysis. Samples were analyzed with a Zeiss Axioplan 2 microscope (Carl Zeiss, Inc, Thornwood, NY) equipped with epifluorescence filters using a 63x magnification lens. Images were captured with a Sony DXC-950P 3CCD camera using Eclipse Imaging software and the extent of uptake was assessed by comparing the numbers of extracellular conidia (labeled with both GFP and the anti-A. fumigatus antibody, hence green and red) to intracellular conidia (green only) after merging the green and red channels in Adobe Photoshop (Adobe, Inc, San Jose, CA). Eight random fields for each coverslip were counted. Each condition for each cell type was performed in triplicate and the data are reported as the mean ± the standard deviation of three independent experiments. The Student's t-test was used to assess the difference between the sialidase-treated and untreated conditions. Confocal images were obtained on a Zeiss LSM 410 confocal microscope equipped with a Krypton/Argon laser 488, 568 and 647 nm lines. Confocal images were processed using ImageJ software (Abramoff et al. 2004).
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Conflict of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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None declared.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsConflict of interest statementAcknowledgmentsReferences |
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We would like to thank Linda Pinto for valuable technical assistance throughout the study and Christine Carson of the Simon Fraser University Microscopy and Imaging Service for assistance with imaging. We would like to thank Denise McDougal of the British Columbia Cancer Agency, Vancouver, Canada for her help with the flow cytometric analysis. We are grateful to the Natural Sciences and Engineering Research Council of Canada and the British Columbia Lung Association for financial support.
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Abbreviations |
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ATCC, American Type Culture Collection; BSA, bovine serum albumin; BSM, bovine submaxillary mucin; DMB, 1,2-diamino-4,5-methylenedioxybenzene, dihydrochloride; ECM, extracellular matrix; FCS, fetal calf serum; GAG, glycosaminoglycan; GFP, green fluorescent protein; HPLC, high-pressure liquid chromatography; IA, invasive aspergillosis; KDN, 2-keto-3-deoxy-nonulosonic acid; MAA, Maackia amurensis agglutinin; MALDI, matrix assisted laser desorption ionization; Neu5Ac, N-acetylneuraminic acid; Neu5,9Ac2, 9-O-acetyl-N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid; PBS, phosphate-buffered saline (pH 7.4); PBS-T, phosphate-buffered saline (pH 7.4)–0.05%Tween 20; PNA, Arachis hypogaea agglutinin; Siglec, sialic acid-binding immunoglobulin-like lectin; SNA, Sambucus nigra agglutinin.
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