Apoptotic machinery in plasmodium falciparum Growing in a continuous culture

Abstract

Apoptosis and indeed metacaspase have not been sufficiently evaluated to show their role in Plasmodium Jalciparum. To study apoptosis, synchronized P. Jalciparum cultures were initiated at a parasitemia of 0.5 % at 5 % hematocrit and allowed to grow in conditions that were not limiting for nutrients and red blood cells until the parasites crashed. Parasite growth was monitored by making Giemsa-stained thin smears that were examined microscopically and by flow cytometry following staining of cells with SYBR green. The following apoptotic processes were evaluated at every stage of the growth curve: DNA fragmentation by TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate assay), mitochondria membrane potential collapse by TMRE (tetra methyl rhodamine ester dye). Expression of metacaspse gene was evaluated by RT-PCR and blotted membranes were probed with polyclonal antibodies for human caspases. P. falciparum initiated at 0.5% ring-stage parasitemia doubled every 48 hours to 1.63 % parasitemia, but the rate of growth declined at 3.68 % and 4%. Consistently, the culture then crashed at about 6% parasitemia when the growth requirements were not limiting. At lower parasite density the malaria parasites were morphologically healthy, but thereafter, the proportion of healthy parasites declined. DNA fragmentation as indicated by TUNE,L positive staining increased from 0.2 % at early parasitemia to 5 % by the time the culture crashed. TMRE staining of the mitochondria showed collapse of mitochondrial membrane potential as the parasite density increased. By qRT-PCR, expression of metacaspase gene was not evident in the ring stages until the parasite density approached 4%. The metacaspase gene was present in trophozoites at 0.5% and 4% parasitemia while in the schizonts it was present at all parasitemia levels. Western blot analysis by anti-caspase 3 and 7 revealed presence of 45 and 28 KDa fragments as it has been reported in other studies. Unlike the metacaspase gene, the protein expression was observed in all the stages. In the ring stages, the protein expression was highest at parasite density of 1.36 % and lowest during the crash. The protein expression pattern was similar in the schizont stages, while at trophozoites stage protein abundance increased as the density increased. Taken .together, these results indicate existence of apoptotic machinery in P. falciparum that seem to operate in tandem with parasite density. These findings offer important insights into P. falciparum survival strategies that could open new avenues for designing rational therapeutic interventions for malaria.