Analysis of Acanthamoeba polyphaga surface carbohydrate exposure by FITC–lectin binding and fluorescence evaluation

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 Kd 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 Bmax, 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.