Lectin Analysis of Trypanosoma congolense Bloodstream Trypomastigote and Culture Procyclic Surface Saccharides by Agglutination and Electron Microscopic Technics*
Authors: JACKSON PETER R. , HONIGBERG B. M. , HOLT STANLEY C. 
Published: May 2007

SYNOPSIS. Living, intact bloodstream trypomastigotes and culture procyclic forms of Trypanosoma congolense were tested for agglutination with the lectins concanavalin A (Con A), phytohemagglutinin P (PP), wheat germ agglutinin (WGA), soybean agglutinin (SBA), and fucose binding protein (FBP). Similar experiments were conducted with living bloodstream and culture forms treated with trypsin or dextranase. Parasites were incubated for 30 min at 25 C in various concentrations of each lectin, then examined for agglutination by dark‐field microscopy. Control preparations consisted of parasites incubated alone or with 0.5 M of the specific competing sugar, with or without the corresponding lectin.

Electron‐microscopic localization of lectin binding sites on the surface of intact and dextranase‐treated bloodstream and intact culture forms was accomplished with Con A, reacted with horseradish peroxidase (HRP) and then diaminobenzidine (DAB). In addition, FBP and SBA were coupled to HRP, then utilized for the localization of binding saccharides on the surface of blood‐stream forms by the DAB technic. Similar studies were conducted with culture procyclics incubated with WGA‐, SBA, PP‐ or FBP‐HRP conjugates and then reacted with DAB. Controls were utilized to confirm the sugar specificity of all positive reactions.

Intact living bloodstream forms were agglutinated in a concentration‐dependent manner with all the lectins tested. Agglutination levels were scored as Con A > FBP > WGA = PP = SBA. Sugars resembling α‐D‐mannose, N‐acetyl‐D‐glucosamine, N‐acetyl‐D‐galactosamine, and α‐L‐fucose are evidently present on the surface of the parasites. No agglutination was noted in any control preparations. Identical lectin‐induced agglutinations were obtained with trypsin‐ or dextranase‐treated bloodstream forms. Trypsin disrupted but did not entirely remove the surface coat of bloodstream forms, while dextranase did not alter the ultrastructure of the parasites. Con A‐, SBA‐ and FBP‐binding saccharides were distributed uniformly on the surface coat of intact bloodstream forms; a similar distribution of Con A receptors was noted also on the surface of dextranase‐treated cells. No lectin‐binding saccharides were visualized by electron microscopy on any control preparations. Intact, trypsin‐ or dextranasetreated, procyclics were agglutinated in a concentration‐dependent fashion by Con A and WGA, but not by the other lectins tested. Control preparations did not agglutinate and the enzymes did not affect the ultrastructure of the parasites. Con A‐ and WGA‐specifically binding saccharides were uniformly distributed on intact procyclics and control preparations were lectin‐negative. Thus, T. congolense procyclics retained surface saccharides resembling α‐D‐mannose and N‐acetyl‐D‐glucosamine but lost sugars resembling N‐acetyl‐D‐galactosamine (or D‐galactose) and α‐L‐fucose. The failure of dextranase to remove the lectin‐binding saccharides from the surface of bloodstream and procyclic forms suggests that α‐1,6‐glucan bonds do not link these carbohydrates. The results are contrasted with lectin research on other trypanosome species and discussed with relation to the biology of T. congolense.