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

Alexander J. Foster, Roger A. Bird, Steven L. Kelly, Kazuko Nishimura, David Poyner, Stephen Taylor, Stephen N. Smith*

*Corresponding author for this work

Research output: Contribution to journalArticle

Abstract

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 a-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.
Original languageEnglish
Pages (from-to)1-8
Number of pages8
JournalMycologia
Volume96
Issue number1
DOIs
Publication statusPublished - 26 Feb 2004

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Cryptococcus neoformans
Fluorescein-5-isothiocyanate
Lectins
lectins
Cell Wall
cell walls
concanavalin A
fluorescence
carbohydrate
cells
ligand
Fluorescence
mannose
Carbohydrates
carbohydrates
microscopy
Mannose
Ligands
Confocal Microscopy
flow cytometry

Keywords

  • confocal microscopy
  • crytococcus
  • FITX-lectins
  • flow cytometry

Cite this

Foster, A. J., Bird, R. A., Kelly, S. L., Nishimura, K., Poyner, D., Taylor, S., & Smith, S. N. (2004). FITC-lectin avidity of Cryptococcus neoformans cell wall and capsular components. Mycologia, 96(1), 1-8. https://doi.org/10.1080/15572536.2005.11832989
Foster, Alexander J. ; Bird, Roger A. ; Kelly, Steven L. ; Nishimura, Kazuko ; Poyner, David ; Taylor, Stephen ; Smith, Stephen N. / FITC-lectin avidity of Cryptococcus neoformans cell wall and capsular components. In: Mycologia. 2004 ; Vol. 96, No. 1. pp. 1-8.
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abstract = "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 a-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.",
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Foster, AJ, Bird, RA, Kelly, SL, Nishimura, K, Poyner, D, Taylor, S & Smith, SN 2004, 'FITC-lectin avidity of Cryptococcus neoformans cell wall and capsular components', Mycologia, vol. 96, no. 1, pp. 1-8. https://doi.org/10.1080/15572536.2005.11832989

FITC-lectin avidity of Cryptococcus neoformans cell wall and capsular components. / Foster, Alexander J.; Bird, Roger A.; Kelly, Steven L.; Nishimura, Kazuko; Poyner, David; Taylor, Stephen; Smith, Stephen N.

In: Mycologia, Vol. 96, No. 1, 26.02.2004, p. 1-8.

Research output: Contribution to journalArticle

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T1 - FITC-lectin avidity of Cryptococcus neoformans cell wall and capsular components

AU - Foster, Alexander J.

AU - Bird, Roger A.

AU - Kelly, Steven L.

AU - Nishimura, Kazuko

AU - Poyner, David

AU - Taylor, Stephen

AU - Smith, Stephen N.

PY - 2004/2/26

Y1 - 2004/2/26

N2 - 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 a-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.

AB - 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 a-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.

KW - confocal microscopy

KW - crytococcus

KW - FITX-lectins

KW - flow cytometry

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