Identification of homologues of the cell cycle regulators in mature in vitro cultivated plasmodium falciparum gametocytes

Abstract

This study was designed to define putative homologues of known eukaryotic cell cycle regulatory proteins in mature in vitro cultivated Plasmodium Jalciparum gametocytes. Cultures of mature gametocytes (16 day-old) were subjected to centrifugation on a step gradient composed of 80%, 65%, 50%, and 35% Percoll dilutions in incomplete culture medium (ICM), comprising RPMI 1640 medium with L-glutamine, 25 mM HEPES and 50 mglL of hypoxanthine. Morphologically mature gametocytes were isolated from the 35/50% (Fraction 2 (F2)) and the 50/65% (Fraction 3 (F3)) Percoll interphase. F2 had the highest concentration of gametocytes and also showed high exflagellation activity unlike F3 which had fewer gametocytes with only occasional exflagellation observed. Pure preparations of each of the stage II, III, IV (early and late), and V gametocytes of P. Jalciparum were obtained at the 35/50% Percoll interphase after subjecting synchronized maturing gametocyte cultures to the same PercolllICM step gradient centrifugation during the 4th, 7th, 10th, 13th, and 16th day of gametocytogenesis. Synchronization of the cultures was achieved by limiting the number of times that. fresh erythrocytes were added to the cultures prior to gametocyte formation to two. It was al~-oDserved that by varying the Percoll concentrations of the gradient used for gametscyte purification, it was possible to obtain pure and enriched preparations of P. falciparum asexual parasites notably rings, trophozoites and schizonts. Western blots' of whole lysates of prepared from these purified preparation of asexual stages (trophozoites and schizonts), gametocyte developmental stages and gametocytes during gamete formation were probed with rabbit anti-cyclin box polyclonal antibody (IgG). This antibody bound, exclusively, to 97 kDa and 116 kDa proteins in gametocyte lysates. These two proteins gradually increased in intensity during gametocytogenesis and then rapidly decreased in intensity and completely disappeared in about 10-12 minutes after the onset of gametogenesis, that is, prior to exflagellation. It was further observed that the 97 and 116 kDa proteins were only detected in gametocytes which were capable of exflagellating but absent in those which showed only ocassional exflagellation. It was proposed that these two proteins are associated with the processes of exflagellation. The results of this study taken together suggest that the 97 and 116 kDa proteins detected by anti-cyclin box polyclonal antibodies in the gametocytes are probably plasmodial cyclins which may bear functional homology to the eukaryotic cyclins in the control of cell division in P. Jalciparum gametocytes.