Trypanosoma rhodesiense: Antigenicity and immunogenicity of flagellar pocket membrane components

Abstract

A low density membrane fraction, isolated from the bloodstream stage of Trypanosoma rhodesiense and enriched in flagellar pocket membrane, was characterized with regard to antigenicity using antibodies raised against purified flagellar pocket membrane. Mild trypsinolysis of flagellar pocket membrane released two small peptides ( M r = 13–16 × 10 3) separated by chromatofocusing (p I = 6.8 and 5.8) that were antigenic as monitored by fused rocket immunoelectrophoresis. Both of these antigenic peptides were enriched in relative fluorescence when flagellar pocket membrane was prepared from surface labeled (fluorescamine-β-cyclodextrin) trypanosomes, indicating that cleaved peptides were on the external (luminal) side of the flagellar pocket membrane. More extensive release of fluorescamine labeled flagellar pocket membrane components was affected using mild detergent treatment (0.15% Zwittergent 3–12 0.4% Triton X-100), crossed immunoelectrophoresis separating two prominent and a third much fainter antigen. Release of the more anodic of the two prominent antigens was more pronounced after incubation of flagellar pocket membrane with either porcine pancreas phospholipase A 2 or umbilical cord sphingomyelinase. The use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent electroblotting to nitrocellulose also revealed two principal flagellar pocket membrane antigens ( M r ≈ 60 and 66 × 10 3), the latter showing greater release after exposure to sphingomyelinase or phospholipase, compared to mild detergent or 50 m M acetate, pH 5.0. Both antigens were glycoprotein as judged by electroblotting and the use of concanavalin A conjugated horseradish peroxidase as probe. Neither flagellar pocket membrane antigen was found to react with monoclonal antibodies prepared against T. rhodesiense variable surface antigen. The use of flagellar pocket membrane in the presence of Freund's complete adjuvant was found to protect mice against challenge infections with either the CP344 clone or uncloned CT Wellcome isolate of T. rhodesiense.