Investigating the molecular participants of programmed cell death in plamodium falciparum

Vieira, Warren Antonio
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Malaria, a disease resulting from infection by members of the Plasmodium genus, accounted for an estimated 627 000 deaths globally in 2012. The majority of these mortalities were due to P. falciparum infections and thus the species of focus in this study. Due to the rapid emergence of drug-resistant strains, novel avenues for research evaluating parasite survival and population regulation within the human host are now needed. Programmed cell death (PCD) is a well characterised means of self-regulation in metazoans, where a plethora of proteins and signals result in the destruction and/or removal of unnecessary, damaged or dangerous cells. A key protein participant is MDM2 which, via its SWIB/MDM2 domain, binds to the nuclear transcription factor p53 to promote p53 degradation and prevent apoptosis. SWIB/MDM2 domains additionally play key roles in transcription-dependent stress survival. No proven PCD molecular participants for P. falciparum exist but two SWIB/MDM2 homologues (PF3D7_0611400 (PfSWIB) and PF3D7_0518200 (PfMDM2)) and a putative p53 homologue (PF3D7_0522400 (Pfp53)) have been identified by bioinformatics. These were assessed experimentally in this study. Structural features of the SWIB/MDM2 domains of PfMDM2 and PfSWIB, suggested that they are chromatin remodelling factors. The domains were amplified from 3D7 P. falciparum genomic DNA, directionally cloned into the pGEX-4T-2 vector, and used for recombinant GST-fusion protein expression in E. coli. The soluble, tagged, domains were isolated and purified by affinity chromatography (PfMDM2, ~33kDa and PfSWIB, ~42kDa) and used, in conjunction with P. falciparum phage display library technology, for the identification of several novel binding partners. Two of these interactions were verified with in vitro binding assays, proving concentration dependent interactions between PfMDM2 and a conserved protein of unknown function; and PfSWIB and a putative Investigating the Molecular Participants of Programmed Cell Death in Plasmodium falciparum serine-threonine protein kinase (PfARK3). Transgenic P. falciparum parasites were created by transfection with pARL2-GFP vector constructs containing the PfSWIB and PfMDM2 genes. PfMDM2-GFP localized to the mitochondria under the control of an N-terminal signal sequence, under normal and heat stress conditions, the latter triggering PCD in the asexual intraerythrocytic parasite. PfSWIB-GFP localized to the cytoplasm under normal and heat stress conditions, but in a subpopulation of trophozoites it moved to the nucleus after exposure to elevated temperatures. PfMDM2 is hypothesized to play a role within the parasite mitochondrion, although its involvement in PCD is uncertain and may be unconventional, while PfSWIB is suggested to be involved in a stage-specific heat stress response. Pfp53 was found to have a putative DNA binding and tetramerization domain, based primarily on sequence alignments. A recombinant GST-tagged form (~87kDa) of these two domains was expressed in E. coli and purified by affinity chromatography. The ability of the recombinant protein to tetramerize was inconclusive, while in an electromobility shift assay it did not bind to a canonical p53 DNA binding consensus sequence identified in the parasite’s genome. The precise cellular function(s) for this protein requires further evaluation. This study represents the first characterisation of these three P. falciparum proteins. Several novel activities were identified for each and their role in PCD was evaluated by exposing parasites to febrile temperatures, which provided new information regarding heat stress regulation in P. falciparum.
A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfilment of the requirements for the degree of Doctor of Philosophy University of the Witwatersrand, Johannesburg, 2014