The major cell surface glycoprotein procyclin is a receptor for induction of a novel form of cell death in African trypanosomes in vitro

Bloodstream forms (BSF) and procyclic culture forms (PCF) of African trypan osomes were incubated with a variety of lectins in vitro. Cessation of cell division and profound morphological changes were seen in procyclic forms b ut not in BSF after incubation with concanavalin A (Con A): wheat germ aggl utinin and Ricinus communis agglutinin. These lectins caused the trypanosom es to cease division, become round and increase dramatically in size, the l atter being partially attributable to the formation of what appeared to be a large 'vacuole-like structure' or an expanded flagellar pocket. Con A was used in all further experiments. Spectrophotometric quantitation of extrac ted DNA and flow cytometry using the DNA intercalating dye propidium iodide showed that the DNA content of Con A-treated trypanosomes increased dramat ically when compared to untreated parasites. Examination of these cells by fluorescence microscopy showed that many of the Con A-treated cells were mu ltinucleate whereas the kinetoplasts were mostly present as single copies, indicating a disequilibrium between nuclear and kinetoplast replication. Im munofluorescence experiments using monoclonal antibodies (mAb) specific for paraflagellar rod proteins and for kinetoplastid membrane protein-11 (KMP- 11), showed that the Con A-treated parasites had begun to duplicate the fla gellum but that this had only proceeded along part of the length of the cel ls, suggesting that the cell division process was initiated but that cytoki nesis was subsequently inhibited. Tunicamycin-treated wild-type trypanosome s and mutant trypanosomes expressing both high levels of non-glycosylated p rocyclins and procyclin isoforms with truncated N-linked sugars were resist ant to the effects of Con A, suggesting that N-linked carbohydrates on the procyclin surface coat were the ligands for Con A binding. This was support ed by data obtained using mutant parasites created by deletion of all three EP procyclin isoforms, two of which contain N-glycosylation sites, by homo logous recombination. The knockout mutants showed reduced binding of fluore scein-labelled Con A as determined by flow cytometry and were resistant to the effects of Con A. Taken together the results show that Con A induces mu ltinucleation, a disequilibrium between nuclear and kinetoplast replication . and a unique form of cell death in procyclic African trypanosomes and tha t the ligands for Con A binding are carbohydrates on the EP forms of procyc lin. The possible significance of these findings for the life cycle of the trypanosomes in the tsetse fly vector is discussed. (C) 2000 Elsevier Scien ce B.V. All rights reserved.