Plasmodium falciparum: programmed cell death in the erythrocytic stages

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. This thesis is presented as a series of publications and unpublished data. Johannesburg, 2015Plasmodium falciparum is responsible for the majority of global malaria deaths. During the pathogenic blood stages of infection, a rapid increase in parasitaemia threatens the survival of the host before transmission of slow-maturing sexual parasites to the mosquito vector to continue the life cycle. Programmed cell death (PCD) may provide the parasite with the means to control its burden on the host and thereby ensure its own survival. PCD in P. falciparum remains a poorly understood and controversial topic. A gathering body of evidence suggests P. falciparum is capable of PCD, but there are conflicting results regarding the phenotype. This study represents a comprehensive phenotypical characterisation of cell death in intraerythrocytic P. falciparum after various physiologically relevant stress stimuli, including high parasitaemia, heat stress simulating febrile paroxysms, and exposure to natural sunlight. The latter is a novel stimulus for PCD studies in P. falciparum. Biochemical markers of cell death, including DNA fragmentation, mitochondrial dysregulation and phosphatidylserine externalisation on parasitized erythrocytes, were used to provide a holistic description of cell death. Data showed that the combination of cell death markers varied with different stress stimuli and with the developmental stage of the parasite. An apoptosis-like phenotype, characterised by mitochondrial depolarisation, DNA fragmentation and phosphatidylserine externalisation, was suggested after stress from high parasitaemia. Heat stress affected ring stage parasites more severely than previous data suggested and induced an apoptosis-like phenotype. In contrast, late stage parasites showed markers of an autophagic-like cell death, including slight DNA fragmentation, phosphatidylserine externalisation and cytoplasmic vacuolisation. Sunlight exposure induced markers of PCD that included DNA fragmentation preceding mitochondrial hyperpolarisation, but the phenotype was not clear. The paradigm of PCD in P. falciparum is a dynamic and ever-evolving one that will continue to challenge our thinking and understanding of how the world’s deadliest parasitic killer can induce its own death to limit damage on the host. Evidence indicates that P. falciparum undergoes PCD and that the phenotype(s) may be unique. PCD is an important feature of P. falciparum biology and the elucidation of parasite PCD pathway(s) that differ from host mechanisms may yield novel drug targets

On Programmed Cell Death in Plasmodium falciparum: Status Quo

Conflicting arguments and results exist regarding the occurrence and phenotype of programmed cell death (PCD) in the malaria parasite Plasmodium falciparum. Inconsistencies relate mainly to the number and type of PCD markers assessed and the different methodologies used in the studies. In this paper, we provide a comprehensive overview of the current state of knowledge and empirical evidence for PCD in the intraerythrocytic stages of P. falciparum. We consider possible reasons for discrepancies in the data and offer suggestions towards more standardised investigation methods in this field. Furthermore, we present genomic evidence for PCD machinery in P. falciparum. We discuss the potential adaptive or nonadaptive role of PCD in the parasite life cycle and its possible exploitation in the development of novel drug targets. Lastly, we pose pertinent unanswered questions concerning the PCD phenomenon in P. falciparum to provide future direction

Nowhere to hide : interrogating different metabolic parameters of Plasmodium falciparum gametocytes in a transmission blocking drug discovery pipeline towards malaria elimination

BACKGROUND : The discovery of malaria transmission-blocking compounds is seen as key to malaria elimination strategies and gametocyte-screening platforms are critical filters to identify active molecules. However, unlike asexual parasite assays measuring parasite proliferation, greater variability in end-point readout exists between different gametocytocidal assays. This is compounded by difficulties in routinely producing viable, functional and stage-specific gametocyte populations. Here, a parallel evaluation of four assay platforms on the same gametocyte populations was performed for the first time. This allowed the direct comparison of the ability of different assay platforms to detect compounds with gametocytocidal activity and revealed caveats in some assay readouts that interrogate different parasite biological functions. METHODS : Gametocytogenesis from Plasmodium falciparum (NF54) was optimized with a robust and standardized protocol. ATP, pLDH, luciferase reporter and PrestoBlue® assays were compared in context of a set of 10 reference compounds. The assays were performed in parallel on the same gametocyte preparation (except for luciferase reporter lines) using the same drug preparations (48 h). The remaining parameters for each assay were all comparable. RESULTS : A highly robust method for generating viable and functional gametocytes was developed and comprehensively validated resulting in an average gametocytaemia of 4 %. Subsequent parallel assays for gametocytocidal activity indicated that different assay platforms were not able to screen compounds with variant chemical scaffolds similarly. Luciferase reporter assays revealed that synchronized stage-specific gametocyte production is essential for drug discovery, as differential susceptibility in various gametocyte developmental populations is evident. CONCLUSIONS : With this study, the key parameters for assays aiming at testing the gametocytocidal activity of potential transmission blocking molecules against Plasmodium gametocytes were accurately dissected. This first and uniquely comparative study emphasizes differential effects seen with the use of different assay platforms interrogating variant biological systems. Whilst this data is informative from a biological perspective and may provide indications of the drug mode of action, it does highlight the care that must be taken when screening broaddiversity chemotypes with a single assay platform against gametocytes for which the biology is not clearly understood.South African Medical Research Council Strategic Health Initiatives Partnerships with the Medicines for Malaria Venture as well as the Council for Scientific and Industrial Research, and the 3R Foundation (project 118–10).http://www.malariajournal.comhb201