Department of Molecular Biology and Biochemistry1 and Department of Biological Sciences2, Simon Fraser University, Burnaby, B.C., Canada V5A 1S6
Author for correspondence: Margo M. Moore. Tel: +1 604 291 3441. Fax: +1 604 291 3496. e-mail: mmoore{at}sfu.ca
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: Aspergillus fumigatus, invasive aspergillosis, lectin, flow cytometry, adhesion
Abbreviations: HPTLC, high-performance thin-layer chromatography; LFA, Limax flavus agglutinin; MAA, Maackia amurensis agglutinin; MALDI, matrix-assisted laser desorption ionization; Neu5Ac, N-acetylneuraminic acid; Neu5Gc, N-glycolylneuraminic acid; SNA, Sambucus nigra agglutinin
a Present address: Department of Microbiology and Immunology, University of British Columbia, Vancouver, B.C., Canada.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The frequency of invasive aspergillosis has increased 14-fold in the past decade (Groll et al., 1996 ), mainly due to the greater numbers of bone marrow and organ transplant recipients (Wald et al., 1997
), more aggressive cytotoxic chemotherapy used to treat cancer patients (Bodey et al., 1992
) and the emergence of the human immunodeficiency syndrome (HIV-AIDS) (Minamoto et al., 1992
). There are currently only two antifungal agents used to treat the disease, amphotericin B and itraconazole, and their success rate is only 34%, even when used prophylactically (Denning, 1998
). The increase in fungal infections worldwide and the lack of satisfactory antifungal therapy makes it critical to understand the mechanisms by which Aspergillus species adhere to and colonize the lung tissue.
Previous work in our laboratory has demonstrated that A. fumigatus conidia bind significantly better than those of other Aspergillus species to intact lung cell basal lamina and fibronectin (Wasylnka & Moore, 2000 ). Investigation of the mechanism of conidial binding determined that negatively charged carbohydrates occurring on the conidial surface may play a role in the adhesion of the conidia to host basal lamina (Wasylnka & Moore, 2000
).
Sialic acids are members of a family of 9-carbon carboxylated monosaccharides that are the major ionogenic compounds in many cell types (Schauer, 1982 ). All compounds are thought to be derived from N-acetylneuraminic acid (Neu5Ac) with the most common modifications being the oxidation of the N-acetyl group to form N-glycolylneuraminic acid (Neu5Gc) and acetylation of the hydroxyl groups at the 4, 7, 8 or 9 position (Varki, 1992
). In most glycoconjugates, the sialic acids are terminal or subterminal non-reducing sugars and are attached by 2,3-
or 2,6-
linkages to underlying galactose residues.
Sialic acids have been shown to be important in several aspects of microbial pathogenesis. Many viruses bind to their host via sialic acids on the epithelial cell surface. For example, influenza A and B viruses recognize sialic acid whereas influenza C viruses bind exclusively to 9-O-acetylated sialic acids (Varki, 1997 ). Incorporation of sialic acids into the capsules of some pathogenic bacteria aids in evading host defences by inhibiting the direct activation of the alternative complement pathway (Wessels et al., 1992
). More recently, sialic acids have been identified in the cell walls of several pathogenic fungi including Candida albicans (Soares et al., 2000
), Cryptococcus neoformans (Rodrigues et al., 1997
), Paracoccidioides brasiliensis (Soares et al., 1998
) and, to a lesser degree, Pneumocystis carinii (De Stefano et al., 1990
). In pathogenic fungi, the biological function of these carbohydrates has not yet been determined but several studies have provided evidence that, as in other microbial pathogens, sialic acids may play a role in host recognition and in the evasion of host defences. For example, one report found that removal of sialic acids from Cry. neoformans yeast cells enhanced their susceptibility to phagocytosis by mouse macrophages (Rodrigues et al., 1997
). The authors postulated that these sialic acids might give the fungus a protective advantage in the host during the early stages of infection (Rodrigues et al., 1997
).
Preliminary analysis of the carbohydrate structures on the conidia of A. fumigatus has shown the presence of D-galacto-D-mannans (Barreto-Bergter et al., 1981 ). Sialic acid substitution was not detected by these investigators because of the conditions used in the isolation procedures. Therefore, the aims of this study were: 1, to determine whether A. fumigatus conidia possess sialoglycoconjugates; 2, to identify the nature of the glycan linkages; and 3, to determine whether the A. fumigatus cell wall contains greater amounts of sialic acid than the cell walls of three non-pathogenic Aspergillus species.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sialidase treatment.
Conidia were harvested from a mature plate of A. fumigatus ATCC 13073 and 2x107 conidia were resuspended in 50 mM sodium acetate buffer, pH 5·0 containing 0 or 1·1 U Clostridium perfringens 2,3--, 2,6-
-, 2,8-
-sialidase (Calbiochem) and incubated for 6 h at 37 °C.
Biotinylation of lectins.
LFA (Calbiochem), S. nigra agglutinin (SNA; Sigma) or M. amurensis agglutinin (MAA; Sigma) (all 1 mg) were incubated with a 20 molar excess of n-hydroxysuccinimidobiotin long chain (Pierce) in 1 ml 0·1 M sodium phosphate buffer, pH 7·4 containing 0·15 M NaCl, for 60 min at room temperature. The biotinylated lectins were then dialysed against PBS in a 4 ml Ultrafree membrane filter with an Mr 10000 cut-off membrane (Millipore).
Lectin labelling.
Sialidase-treated samples.
Untreated or sialidase-treated A. fumigatus conidia were reacted with biotinylated LFA essentially as described by Rodrigues et al. (1997) with the following modifications. Cells were blocked for 60 min with PBS/10% goat serum and incubated with biotinylated LFA at 50 µg ml-1 in PBS/10% goat serum for 60 min; bound lectin was detected by incubation for 60 min with StreptavidinOregon Green (Molecular Probes) diluted 1:150 in PBS/10% goat serum. Flow cytometry was performed on a Coulter EPICS Elite Esp flow cytometer using a 488 nm laser for excitation energy and a 550 nm dichroic filter capturing an emission band at 525 nm: 10000 events were recorded for each sample. Samples were also viewed with an Olympus VANOX AHBS3 microscope equipped with epifluorescence at 1000x magnification. Bright field and fluorescent images for each sample were captured with a Sony 950 camera using Eclipse (Empix Imaging) image capturing software.
Determination of sialic acid linkage.
To determine the sialic acid linkages in A. fumigatus conidia, an aliquot of 2x107 cells was lectin labelled as described above using 50 µg biotinylated LFA, SNA or MAA ml-1 and then analysed by flow cytometry or fluorescence microscopy as described above.
Competition of LFA binding by sialic acid.
An aliquot of 3·6x107 A. fumigatus ATCC 13073 conidia was added to poly L-lysine coated coverslips for 60 min and reacted with biotinylated LFA in PBS/5% goat serum containing 0 or 100 mM Neu5Ac (Rose Scientific). The remainder of the protocol was as described above. Samples were mounted with ProLong mounting media (Molecular Probes) and analysed by fluorescence microscopy.
Lectin labelling of different Aspergillus species.
Conidia from A. fumigatus ATCC 13073, ATCC 42202 and CHUV; A. ornatus ATCC 16921 and ATCC 66492; A. wentii ATCC 10584 and ATCC 1023; and A. auricomus ATCC 16890 were collected, and 1x107 conidia were labelled with biotinylated LFA and processed for flow cytometry analysis as described above.
Isolation of sialic acids from A. fumigatus conidia.
Sialic acids from A. fumigatus conidia were isolated as described by Reuter & Schauer (1994) with minor modifications. Briefly, dry cells (5·5 g) were resuspended in 2030 ml 88% formic acid for 60 min at 70 °C. The suspension, which should contain any O-acetylated sialic acids, was cooled on ice and centrifuged for 10 min at 1500 g. The supernatant was removed, and the pellet resuspended in 2030 ml 3 M HCl and incubated at 80 °C for 60 min to remove all remaining sialic acids. This suspension was cooled and centrifuged as described above. The supernatants from both the formic and hydrochloric acid digests were then ultracentrifuged for 30 min at 50000 g and 4 °C, and the ultrasupernatants were lyophilized overnight. The dried residues were resuspended in 4 ml cold, deionized, distilled H2O (ddH2O) and then dialysed against ddH2O in a 5000 Da (HCl digest) or 10000 Da (HCOOH digest) molecular mass cut-off Ultrafree-4 centrifugal filter (Millipore). The diffusate was passed over a Dowex 50WX8 (100200 mesh) cation exchange column (hydrogen ion form; Sigma) and washed with 34 bed vols cold ddH2O. The eluate and washings were collected, lyophilized, resuspended in 5 ml cold ddH2O and the pH adjusted to 4·0 with NH4OH. This solution was then passed over a Dowex 2X8 (200400 mesh) anion exchange column (formate form; Sigma), washed with 10 ml cold ddH2O and then eluted with 20 ml 1 M formic acid. Fractions of 1·5 ml were collected and lyophilized. The dried residues were resuspended in 40 µl ddH2O and 10 µl was spotted onto HPTLC (high-performance thin-layer chromatography) plates (Mandel Scientific) along with 5 µl Neu5Ac and Neu5Gc (Sigma) standards (at 1 mg ml-1) and run in 1-propanol/1 M NH3/H2O (6:2:1 by vol.). Plates were developed by spraying with orcinol/HCl spray reagent (81·4 ml 37% HCl, 0·2 g orcinol (Sigma), 2 ml 1% FeCl3) diluted 3:1 with ddH2O and then heated at 180 °C for 20 min.
Colorimetric quantitation of sialic acids.
For colorimetric quantitation, 4x1010 conidia were hydrolysed with 0·1 M trichloroacetic acid for 3 h at 80 °C. The suspension was cooled and centrifuged at 9000 g for 10 min. The supernatant was then treated with 0·1 M HCl for 1 h at 80 °C and dialysed overnight in a 3500 Da dialysis cassette (Pierce) (4 °C, 750 ml ddH2O, two changes). The next day the diffusate was concentrated by rotary vacuum until the final volume was 250 ml and this was lyophilized to dryness. Ion-exchange chromatography was performed as described above. To further purify the sialic acids from contaminants, anion exchange fractions were streaked onto HPTLC plates and the sialic acid containing bands scraped off and extracted with ddH2O followed by 1:1 ddH2O/methanol. These extracts were lyophilized by speed vacuum, resuspended in ddH2O and analysed by colorimetric quantitation as described by Reuter & Schauer (1994) by measuring the absorbance at 572 nm of samples reacted with the orcinol/Fe3+/HCl reagent.
Matrix-assisted laser desorption ionization (MALDI) MS.
Mass spectra of purified sialic acids were obtained using a MALDI mass spectrometer from PerSeptive Biosystems with an accelerating voltage of 20 keV. An aliquot of purified sialic acids (2·5 µl) was mixed with 0·5 µl matrix (10 mg 2,5-dihydroxybenzoic acid ml-1) and laser ionization focused around areas of good crystallization.
Adhesion to poly L-lysine.
Sialidase-treated or untreated A. fumigatus conidia were added to poly L-lysine (Sigma) coated coverslips in 24 well plates at a concentration of 1x107 conidia ml-1 for 60 min at room temperature. Conidia were fixed with PBS/4% (w/v) paraformaldehyde, pH 7·4 for 60 min and then coverslips were mounted onto slides. Samples were viewed with an Olympus Vanox AHBS3 microscope at a 1000x magnification. Sample enumeration was performed by collecting images from five random fields (Sony 950 video camera) and the number of conidia per field was calculated by the computer program Eclipse.
Adhesion assays.
Experiments were performed as described previously (Wasylnka & Moore, 2000 ). Mucin (Type I-S, Sigma), asialomucin or sialic acid were co-incubated with peroxidase-labelled conidia at the indicated concentrations.
Miscellaneous chemicals and supplies.
Vibrio cholerae sialidase was obtained from Calbiochem and Arthrobacter ureafaciens sialidase was from Sigma. All chemicals were reagent grade or better unless specifically noted. Asialomucin was a generous gift from Dr J. Esko (Division of Cellular and Molecular Medicine, University of California, San Diego, CA, USA).
Statistics.
Differences in sialic acid densities between Aspergillus species were analysed by a one-way KruskalWallis test for non-parametric data using JMP (SAS Institute). The Students t test was used for statistical analysis of other data. Values shown are mean data from three independent experiments (unless otherwise stated). A P value of <0·05 was considered significant.
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
A. fumigatus conidia were incubated with biotinylated LFA lectin, which reacts with any sialic acid linkage, (Schauer et al., 1995 ) and then with a secondary fluorophore, StreptavidinOregon Green. A. fumigatus conidia were strongly fluorescent (compared to the sample which received StreptavidinOregon Green only), indicating the presence of sialic acids on the conidial surface (Fig. 1a
). Analysis by flow cytometry (Fig. 1
) and epifluorescence (Fig. 2ad
) of sialidase-treated and untreated conidia showed that pre-treatment of conidia with Clo. perfringens sialidase significantly reduced the fluorescent signal. Three independent experiments using flow cytometry showed that sialidase-treated conidia had 48±18% of the mean fluorescence when compared to the controls (set at 100%). We found that removal of sialic acids was only achieved at high concentrations of the Clo. perfringens sialidase (1·1 U). Lower concentrations of Clo. perfringens sialidase, V. cholerae sialidase or A. ureafaciens sialidase were ineffective at cleaving the sialic acids (data not shown).
|
|
To determine the glycosidic linkage of the sialic acids, we reacted A. fumigatus conidia with lectins that bind specifically to different sialic acid linkages. Conidia were incubated with biotinylated LFA, MAA or SNA. MAA reacts with Neu5Ac2,3--Gal1,4-ß-GlcNAc and SNA reacts with Neu5Ac2,6-
-Gal/GalNAc (Schauer et al., 1995
). The extent of MAA and SNA binding to A. fumigatus conidia was much lower than that observed with LFA: LFA-treated conidia were 13 times more fluorescent than SNA-treated conidia and 16 times more fluorescent than MAA-treated conidia as determined by flow cytometry (Fig. 3
). To confirm that both SNA and MAA were functional and adequately biotinylated, the lectins were used as probes for binding to sialylated glycoproteins fetuin (MAA) and fibronectin (SNA) immobilized onto nitrocellulose. Both SNA and MAA reacted equally with these glycoproteins (data not shown), confirming that the lack of reactivity was due to the structure of the conidial carbohydrates. Since 2,6-
-sialic acid has been the predominant sialic acid structure identified in other pathogenic fungi (Rodrigues et al., 1997
; Soares et al., 1998
, 2000
), the lack of reactivity with SNA was surprising. However, filamentous fungi such as Aspergillus may have novel sialic acids that are not found in yeast-like fungi such as Candida and Cryptococcus. In summary, the lack of reactivity of A. fumigatus conidia with SNA and MAA suggests that 2,3-
- or 2,6-
-linked sialic acids represent a minority of the sialic acid structures present on the A. fumigatus conidial cell walls. Alternatively, the sialic acid may be substituted in such a way that reduces SNA or MAA binding or that other sialic acid linkages, in particular 2,8-
or 2,9-
, may be present in A. fumigatus conidia. However, since there are no lectins that bind to these structures, this cannot be proven without a more detailed chemical analysis of purified sialic acids.
|
|
|
|
Conidia of pathogenic Aspergillus species possess greater amounts of sialic acid than non-pathogenic species
Previous work in our laboratory has shown that A. fumigatus conidia bind to purified fibronectin and intact basal lamina significantly better than other non-pathogenic Aspergillus species (Wasylnka & Moore, 2000 ). The biological function of sialic acids in pathogenic fungi is still unknown; however, they are important virulence factors in other micro-organisms such as T. cruzi (Burleigh & Andrews, 1995
) and Neisseria meningitidis (Vogel et al., 1996
). To determine whether the density of sialic acids may be correlated with adhesion to basal lamina in Aspergillus, we measured the density of sialic acid on conidia from four species of Aspergillus. In addition to three strains of A. fumigatus, three non-pathogens were also studied: A. wentii (two strains), A. ornatus (two strains) and A. auricomus (one strain). A. fumigatus is the most common cause of invasive aspergillosis (Denning, 1998
), while A. wentii, A. ornatus and A. auricomus have never been documented in human disease (Pitt, 1994
). Adhesion to basal lamina has been previously measured in three of these four species (Wasylnka & Moore, 2000
).
Conidia from all four Aspergillus species were collected from agar plates and incubated with biotinylated LFA followed by StreptavidinOregon Green. Analysis by fluorescence microscopy provided preliminary evidence that all three A. fumigatus strains contained greater amounts of sialic acids than the other Aspergillus species tested (data not shown). Therefore, the experiment was repeated and the conidia analysed by flow cytometry to quantitatively measure sialic acid density in all four species. Because differences in the shape and size of conidia can affect the data spread in flow cytometry, each species was labelled with both LFA and StreptavidinOregon Green (sample fluorescence) and also with StreptavidinOregon Green alone as a control. By dividing the mean of the control fluorescence by the value of the sample fluorescence, a mean sample value was obtained which enabled a direct comparison between species. The mean fluorescence of A. fumigatus ATCC 42202 and CHUV was four times greater than the A. auricomus strain, 10 times greater than both A. ornatus strains and the A. wentii ATCC 1023 strain and 20 times greater than A. wentii ATCC 10584 (Table 1). The mean fluorescence of the other A. fumigatus strain, ATCC 13073, was three times greater than the A. auricomus strain, six times greater than both A. ornatus strains and the A. wentii ATCC 1023 strain and 13 times greater than A. wentii ATCC 10584 (Table 1
). Statistical analysis of the mean fluorescence using the KruskalWallis test demonstrated that the mean fluorescence of all the A. fumigatus strains was significantly greater than any of the non-A. fumigatus strains (P<0·05). Therefore, these experiments showed that the expression of sialic acids on A. fumigatus conidial walls was 320 times greater than the conidia of the three non-pathogenic Aspergillus species. In addition, the flow cytometry data comparing sialic acid expression between Aspergillus species parallels our data on the adhesion of Aspergillus species to basal lamina: all A. fumigatus strains bound to basal lamina with 1020 fold greater affinity than A. ornatus and A. wentii strains (A. auricomus was not tested) (Wasylnka & Moore, 2000
). Taken together, these data indicate a correlation between sialic acid density and adhesion to basal lamina proteins.
|
Role of conidial sialic acid in the binding of A. fumigatus to fibronectin and basal lamina
To confirm that the observed differences in sialic acid density between species correlated with adhesion to basal lamina, we tested the adhesion of A. fumigatus conidia to components of the basal lamina in the presence of sialylated carbohydrates. We used this method because, as stated earlier, sialidase treatment of conidia removes only 50% of the sialic acids from the spore surface and requires large amounts of enzyme. We first investigated the adhesion of A. fumigatus conidia to purified fibronectin. Conidia were co-incubated with the heavily sialylated glycoprotein mucin (12% sialic acid by weight), asialomucin or sialic acid and allowed to adhere to fibronectin-coated microtitre plates. Binding of conidia to immobilized fibronectin was inhibited almost completely (by 85%) by 250 µg mucin ml-1 and only partially (62%) by asialomucin (Fig. 7). Furthermore, at 125 µg ml-1, mucin still inhibited binding by 56%, whereas binding to fibronectin in the presence of asialomucin was essentially restored. Therefore, mucin had a dose-dependent inhibitory effect on conidia binding to fibronectin. The inability of asialomucin to strongly reduce binding suggests that it is the sialic acid residues on mucin which are directly involved in the inhibitory effect. Further evidence for the involvement of sialic acids in binding to fibronectin was demonstrated using sialic acid as an inhibitor in binding assays. Co-incubation of conidia with 20 mM sialic acid inhibited binding by 90% (data not shown), although part of the reduction in binding may have been due to the acid pH of the sialic acid solution (pH 3·5 at 20 mM).
|
We next investigated whether sialic acids on A. fumigatus conidia were involved in binding to immobilized basal lamina. Conidia were again co-incubated in the presence of mucin, asialomucin or sialic acid and allowed to adhere to basal-lamina-coated microtitre plates. In contrast to the results obtained with fibronectin, mucin (up to 500 µg ml-1) had no effect on binding of conidia to basal lamina (data not shown). However, as seen with fibronectin, sialic acid was also able to strongly inhibit binding to basal lamina (69% at 20 mM) (data not shown). Therefore, in contrast to fibronectin, the binding to intact basal lamina may be more complex; perhaps requiring the participation of surface sialic acids and/or other negatively charged carbohydrates on the spore surface. Nonetheless, it has been suggested that the basal lamina of diseased lungs have increased amounts of fibronectin and other extracellular matrix proteins (Roman & McDonald, 1997 ). Therefore, binding of A. fumigatus conidia to fibronectin in vivo may be important in the development of invasive aspergillosis.
![]() |
ACKNOWLEDGEMENTS |
---|
The authors would like to thank Dr R. Durand (Department of Medical Biophyics, B.C. Cancer Agency, Vancouver, Canada) for use of the Coulter EPICS Elite flow cytometer and helpful discussions, and Denise McDougal for running the samples. The authors also acknowledge Dr B. M. Pinto (Department of Chemistry, Simon Fraser University, Burnaby, Canada) for protocols on the isolation of sialic acids from whole cells.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Barreto-Bergter, E., Gorin, P. A. J. & Travassos, L. R. (1981). Cell constituents of mycelia and conidia of A. fumigatus. Carbohydr Res 95, 205-218.
Bodey, G. P. & Vartivarian, S. (1989). Aspergillosis. Eur J Clin Microbiol Infect Dis 8, 413-437.[Medline]
Bodey, G., Bueltmann, B., Duguid, W. & 11 other authors (1992). Fungal infections in cancer patients: an international autopsy survey. Eur J Clin Microbiol Infect Dis 11, 99109.[Medline]
Bouchara, J. P., Sanchez, M., Chevailler, A., Marot-Leblond, A., Lissitzky, J. C., Tronchin, G. & Chabasse, D. (1997). Sialic acid-dependent recognition of laminin and fibrinogen by Aspergillus fumigatus conidia. Infect Immun 65, 2717-2724.[Abstract]
Broderick, L. S., Conces, D. J.Jr, Tarver, R. D., Bergmann, C. A. & Bisesi, M. A. (1996). Pulmonary aspergillosis: a spectrum of disease. Crit Rev Diagn Imaging 37, 491-531.[Medline]
Burleigh, B. A. & Andrews, N. W. (1995). The mechanisms of Trypanosoma cruzi invasion of mammalian cells. Annu Rev Microbiol 49, 175-200.[Medline]
Denning, D. W. (1998). Invasive aspergillosis. Clin Infect Dis 26, 781-803.[Medline]
De Stefano, J. A., Cushion, M. T., Puvanesarajah, V. & Walzer, P. D. (1990). Analysis of Pneumocystis carinii cyst wall. II. Sugar composition. J Protozool 37, 436-441.[Medline]
Gilbert, M., Bayer, R., Cunningham, A. M., DeFrees, S., Gao, Y., Watson, D. C., Young, N. M. & Wakarchuk, W. W. (1998). The synthesis of sialylated oligosaccharides using a CMP-Neu5Ac synthetase/sialyltransferase fusion. Nat Biotechnol 16, 769-772.[Medline]
Groll, A. H., Shah, P. M., Mentzel, C., Schneider, M., Just-Nuebling, G. & Huebner, K. (1996). Trends in the postmortem epidemiology of invasive fungal infections at a university hospital. J Infect 33, 23-32.[Medline]
Hearn, V. M. & Sietsma, J. H. (1994). Chemical and immunological analysis of the Aspergillus fumigatus cell wall. Microbiology 140, 789-795.[Abstract]
Latge, J. P. (1999). Aspergillus fumigatus and aspergillosis. Clin Microbiol Rev 12, 310-350.
Minamoto, G. Y., Barlam, T. F. & Vander Els, N. J. (1992). Invasive aspergillosis in patients with AIDS. Clin Infect Dis 14, 66-74.[Medline]
Pitt, J. I. (1994). The current role of Aspergillus and Penicillium in human and animal health. J Med Vet Mycol 32, 17-32.[Medline]
Reuter, G. & Schauer, R. (1994). Determination of sialic acids. Methods Enzymol 230, 168-199.[Medline]
Rodrigues, M. L., Rozental, S., Couceiro, J. N., Angluster, J., Alviano, C. S. & Travassos, L. R. (1997). Identification of N-acetylneuraminic acid and its 9-O-acetylated derivative on the cell surface of Cryptococcus neoformans: influence on fungal phagocytosis. Infect Immun 65, 4937-4942.[Abstract]
Roman, J. & McDonald, J. A. (1997). Fibronectins and fibronectin receptors in lung development, injury and repair. In The Lung: Scientific Foundations , pp. 737-755. Edited by R. G. Crystal & J. B. West. Philadelphia, PA: Lippincott-Raven.
Schauer, R. (1982). Chemistry, metabolism and biological function of sialic acids. Adv Carbohydr Chem Biochem 40, 131-234.[Medline]
Schauer, R., Kelm, S., Reuter, G., Roggentin, P. & Shaw, L. (1995). Biochemistry and the role of sialic acids. In Biology of the Sialic Acids , pp. 7-67. Edited by A. Rosenberg. New York: Plenum.
Soares, R. M. A., Costa e Silva-Filho, F., Rozental, S., Angluster, J., de Souza, W., Alviano, C. S. & Travassos, L. R. (1998). Anionogenic groups and surface sialoglycoconjugate structures of yeast forms of the human pathogen Paracoccidioides brasiliensis. Microbiology 144, 309-314.[Abstract]
Soares, R. M., de A. Soares, R. M., Alviano, D. S., Angluster, J., Alviano, C. S. & Travassos, L. R. (2000). Identification of sialic acids on the cell surface of Candida albicans. Biochim Biophys Acta 1474, 262-268.[Medline]
Varki, A. (1992). Diversity in the sialic acids. Glycobiology 2, 25-40.[Medline]
Varki, A. (1997). Sialic acids as ligands in recognition phenomena. FASEB J 11, 248-255.
Vogel, U., Hammerschmidt, S. & Frosch, M. (1996). Sialic acids of both the capsule and the sialylated lipooligosaccharide of Neisseria meningitidis serogroup B are prerequisites for virulence of meningococci in the infant rat. Med Microbiol Immunol 185, 81-87.[Medline]
Wald, A., Leisenring, W., van Burik, J. A. & Bowden, R. A. (1997). Epidemiology of Aspergillus infections in a large cohort of patients undergoing bone marrow transplantation. J Infect Dis 175, 1459-1466.[Medline]
Wasylnka, J. A. & Moore, M. M. (2000). Adhesion of Aspergillus species to extracellular matrix proteins: evidence for involvement of negatively charged carbohydrates on the conidial surface. Infect Immun 68, 3377-3384.
Wessels, M. R., Haft, H. T., Heggen, L. M. & Rubens, C. E. (1992). Identification of a genetic locus essential for capsule sialylation in type III group B streptococci. Infect Immun 60, 392-400.[Abstract]
Received 16 October 2000;
revised 4 January 2001;
accepted 8 January 2001.