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FITC-lectin avidity of Cryptococcus neoformans cell wall and capsular components

     Life and Health Sciences, Aston University, Birmingham, B4 7ET, United Kingdom

Roger A. Bird

     The Medical School, University of Birmingham, Birmingham, B15 2TJ, United Kingdom

Steven L. Kelly

     Wolfson Laboratory of P450 Biodiversity, Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, SY23 3DA, Wales, United Kingdom

Kazuko Nishimura

     Research Centre for Pathogenic Fungi and Microbial Toxicoses, Chiba University, 1–8–1 Inohana, Chuo-ku, Chiba-shi, Chiba, 260–8673 Japan

David Poyner

     Life and Health Sciences, Aston University, Birmingham, B4 7ET, United Kingdom

Stephen Taylor

     Institute of Biological Sciences, University of Wales Aberystwyth, Aberystwyth, SY23 3DA, Wales, United Kingdom

Stephen N. Smith ¹

     Life and Health Sciences, Aston University, Birmingham, B4 7ET, United Kingdom

Flow cytometry and confocal microscopy were used to quantify and visualize FITC-lectin binding to cell-surface carbohydrate ligands of log and stationary phase acapsular and capsular Cryptococcus neoformans strains. Cell populations demonstrated marked avidity for terminal -linked mannose and glucose specific FITC-Con A, mannose specific FITC-GNL, as well as N-acetylglucosamine specific FITC-WGA. Exposure to other FITC-lectins specific for mannose, fucose and N-acetylgalactosamine resulted in little cell-surface fluorescence. The nature of cell-surface carbohydrates was investigated further by measurement of the fluorescence from surfaces of log and stationary phase cell populations after exposing them to increasing concentrations of FITC-Con A and FITC-WGA. Cell fluorescence increased significantly with small increases in FITC-Con A and FITC-WGA concentrations attaining reproducible maxima. Measurements of this nature supported calculation of the lectin binding determinants EC 50, Hn, Fmax and relative Bmax values. EC50 values indicated that the yeast-cell surfaces had greatest affinity for FITC-WGA, however, relative Bmax values indicated that greater numbers of Con A binding sites were present on these same cell surfaces. Hn values suggested a co-operative lectin-carbohydrate ligand interaction. Imaging of FITC-Con A and FITC-WGA cell-surface fluorescence by confocal microscopy demonstrated marked localization of both lectins to cell surfaces associated with cell division and maturation, indicative of dynamic carbohydrate ligand exposure and masking. Some fluorescence was associated with entrapment of FITC-Con A by capsular components, but FITC-Con A and FITC-WGA readily penetrated the capsule matrix to bind to the same cell surfaces labelled in acapsular cells.

Key words: confocal microscopy, Cryptococcus, FITC-lectins, flow cytometry

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