Analysis of Acanthamoeba polyphaga surface carbohydrate exposure by FITC-lectin binding and flourescence evaluation

E.A.G. Elloway, Richard A. Armstrong, R.A. Bird, S.L. Kelly, S.N. Smith*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

Abstract

Aims: Characterization of the representative protozoan Acanthamoeba polyphaga surface carbohydrate exposure by a novel combination of flow cytometry and ligand-receptor analysis. Methods and Results: Trophozoite and cyst morphological forms were exposed to a panel of FITC-lectins. Population fluorescence associated with FITC-lectin binding to acanthamoebal surface moieties was ascertained by flow cytometry. Increasing concentrations of representative FITC-lectins, saturation binding and determination of K d and relative Bmax values were employed to characterize carbohydrate residue exposure. FITC-lectins specific for N-acetylglucosamine, N-acetylgalactosamine and mannose/glucose were readily bound by trophozoite and cyst surfaces. Minor incremental increases in FITC-lectin concentration resulted in significant differences in surface fluorescence intensity and supported the calculation of ligand-binding determinants, Kd and relative B max, which gave a trophozoite and cyst rank order of lectin affinity and surface receptor presence. Conclusions: Trophozoites and cysts expose similar surface carbohydrate residues, foremost amongst which is N-acetylglucosamine, in varying orientation and availability. Significance and Impact of the Study: The outlined versatile combination of flow cytometry and ligand-receptor analysis allowed the characterization of surface carbohydrate exposure by protozoan morphological forms and in turn will support a valid comparison of carbohydrate exposure by other single-cell protozoa and eucaryotic microbes analysed in the same manner.

Original languageEnglish
Pages (from-to)1319-1325
Number of pages7
JournalJournal of Applied Microbiology
Volume97
Issue number6
DOIs
Publication statusPublished - 13 Dec 2004

Keywords

  • acanthamoeba
  • FITC-lectins
  • flow cytometry
  • ligand-receptor analysis
  • surface carbohydrates

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