Molecular and cellular investigations on apoptosis-like cell death in human and animal Plasmodium parasites

Because of emerging resistances against the main antimalarial drugs there is an increasing need for new drugs with alternative targets. As a new target apoptosis can be considered as an appropriate option to attain this goal. There is little information available about programme cell death (PCD) in Plasmodium parasites, and the few we know comes from studies on PCD in P. falciparum and P. berghei. The general objective in this study was increasing the knowledge on P. vivax biology focusing on PCD apoptosis-like processes topic. The specific objectives were: 1- Genetic characterization of two putative PCD genes: Plasmodium vivax apoptosis related protein (PvARP) and of P. vivax meta-caspase 1 (PvMC1). 2- Set-up of P. vivax short term in vitro cultures to study morphological features of PCD. 3- Investigation at the ultra-structural level of morphological aspects of apoptosis-like phenomenon in Plasmodium by using P. falciparum and P. berghei as study model. In this study, Plasmodium vivax apoptosis related protein (PvARP), P. vivax meta-caspase 1 (PvMCA1), P. falciparum meta-caspase 1 (PfMCA1) and P. vivax merozoite surface protein-1(PvMSP1) genes were characterized using PCR method among Plasmodium isolates collected from malaria patients with different origins of the world. PCR products of PvMCA1, PvARP and PfMCA1 genes were sequenced. Also Reverse Transcription-PCRs (RT-PCR) of PvMCA1, PvARP and PvMSP1 genes were performed among thirty frozen blood samples. Analysis of the PvMCA1 gene clearly showed size polymorphism and among isolates analyzed, four different allele sizes were distinguished. Within 30 of the vivax isolates analyzed, four different allele sizes were detected for PvMSP1 gene. Characterization of PfMCA1 gene on thirty falciparum isolates did not show any variations on the basis of molecular length of amplicons. Characterization and sequencing of PvARP gene led to detection of the gene consisting 1975 nucleotides including 7 exons and 6 introns. PvARP amplicons among all analyzed isolates did not show any size variation. Specific RT-PCR products for PvARP and PvMSP1 genes were detected in ten of thirty samples, whereas PvMCA1 analysis amplified fragments only in two samples. To carry out of Transmission Electron Microscopy (TEM) analyses, P. falciparum infected erythrocytes were obtained from continuous cultures. Also P. berghei infected erythrocytes were collected from untreated and treated infected BALB/c mice with Auranofin. Moreover a Short-term in vitro culture was set up for P. vivax and the parasites were treated with staurosporine and chloroquine. Incubation was stopped after 12 and 18-h stimulation. TEM sections were prepared from RBC pellets using specimen preparation process for TEM. Electron micrographs of different stages of P. falciparum and P. berghei were prepared and the different structures of the parasites were specified through comparison with the references. We couldn't obtain TEM photos of P. vivax from the short term culture samples, due to the very low initial parasitemia and the disappearing of parasite in the treated samples. In this study for the first time, size polymorphism in the P. vivax metacaspase 1 was identified. Recently, metacaspase proteins have identified in Plasmodium species and potentially involved in PCD of the malaria parasites. To know more about genetic diversity in vivax populations could provide useful information for the implementation of malaria control activities. We confirmed the presence of 7 exons and 6 introns in PvARP similar to PfARP gene. The absence of RT-PCR products of PvMCA1 in eight samples positively amplified for PvARP and PvMSP1 suggests the hypothesis that probably these results could be associated with expression rate of PvMCA1. In the TEM part of the study, the morphological changes of P. berghei exposed to the Auranofin were explored as a model in the frame of PCD study and structural alterations including vacuolization and parasite cell lysis with leakage of cytosol were observed. Although interesting results has been achieved in TEM samples from treated P. berghei, we have to consider these as preliminary results and more studies are recommended. To reproduce TEM protocol used with falciparum and berghei for P. vivax was hampered by a list of problems reported as follows: 1) The inability to maintain P. vivax in continues cultures. 2) The difficult in recovering viable P. vivax parasites after the preservation in liquid nitrogen 3) The problems occurred during transport of fresh blood samples from endemic area to the equipped laboratory. 4) The problems to find other patients with higher level of parasitemia in an endemic area in Iran. In this study, we succeeded in providing new information about the genetics of P. vivax and we believe that the TEM protocol we set up using P. falciparum and P. berghei could be a useful and crucial tool to study at the ultra-structural level the biology of this still neglected human parasite.