Analysis of Acanthamoeba polyphaga surface carbohydrate exposure by FITC-lectin binding and fluorescence evaluation.
OBJECTIVE
Characterization of the representative protozoan Acanthamoeba polyphaga surface carbohydrate exposure by a novel combination of flow cytometry and ligand-receptor analysis.


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 B(max) 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, K(d) 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.


CONCLUSIONS
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.
Elloway
E A G
EA
Life and Health Sciences, Aston University, Birmingham, UK.
Armstrong
R A
RA
Bird
R A
RA
Kelly
S L
SL
Smith
S N
SN
D000048:Acanthamoeba / Q000378:metabolism*; D000116:Acetylgalactosamine / Q000378:metabolism; D000117:Acetylglucosamine / Q000378:metabolism; D000818:Animals; D050260:Carbohydrate Metabolism; D005434:Flow Cytometry / Q000379:methods; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism*; D005453:Fluorescence; D005947:Glucose / Q000378:metabolism; D037102:Lectins / Q000378:metabolism*; D008024:Ligands; D008358:Mannose / Q000378:metabolism; D013499:Surface Properties
D037102:Lectins; D008024:Ligands; D016650:Fluorescein-5-isothiocyanate; D005947:Glucose; D000116:Acetylgalactosamine; D008358:Mannose; D000117:Acetylglucosamine