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Differences in sialic acid density in pathogenic and non-pathogenic Aspergillus species

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

Aspergillus fumigatus is a ubiquitous soil fungus that causes invasive lung disease in the immunocompromised host. The structure of the conidial wall has not been well characterized although it is thought that adhesins present on the surface are involved in attachment of the conidia to host lung cells and proteins, which is a prerequisite for the establishment of infection. Negatively charged carbohydrates on the conidial surface have been previously identified as the molecules responsible for attachment of conidia to extracellular matrix proteins. The aim of this research was to identify carbohydrates on the conidial surface that contribute to its negative charge. Direct chemical analysis and indirect binding assays have demonstrated that A. fumigatus possesses sialic acids on the conidial surface. Pre-treatment of A. fumigatus conidia with sialidase decreased binding of a sialic acid-specific lectin, Limax flavus agglutinin (LFA), to the conidial surface and decreased adhesion of conidia to the positively charged polymer poly L-lysine. Two other sialic acid-specific lectins, Maackia amurensis agglutinin and Sambucus nigra agglutinin, exhibited negligible binding to A. fumigatus conidia indicating that 2,3-- and 2,6--linked sialic acids are not the major structures found on the conidial surface. Mild acid hydrolysis and purification of conidial wall carbohydrates yielded a product that had the same R_F as the Neu5Ac standard when analysed by high-performance thin-layer chromatography. A density of 6·7x10⁵ sialic acid residues per conidium was estimated using a colorimetric assay. Conidia grown on a minimal medium lacking sialic acid also reacted with LFA, indicating that sialic acid biosynthesis occurs de novo. Sialic acid biosynthesis was shown to be regulated by nutrient composition: the density of sialic acids on the surface of conidia grown in minimal media was lower than that observed when conidia were grown on rich, complex media. It has previously been shown that pathogenic Aspergillus species adhere to basal lamina proteins to a greater extent than non-pathogenic Aspergillus species. To determine whether the expression of sialic acid on the conidial surface was correlated with adhesion to basal lamina, conidia from other non-pathogenic Aspergillus species were tested for their reactivity towards LFA. Flow cytometric analysis demonstrated that A. fumigatus had a significantly greater sialic acid density than three non-pathogenic Aspergillus species. Sialic acids on the conidial wall may be involved in adhesion to fibronectin, a component of the basal lamina, as binding of A. fumigatus conidia to fibronectin was strongly inhibited in the presence of a sialylated glycoprotein.

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.

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