Investigating phenotypic differences of Plasmodium falciparum parasites following exposure to classical antimalarial drugs

Abstract

Malaria remains a leading health problem with an estimated 445 000 deaths occurring in 2016 of which the majority of deaths occurred in Africa. Currently, the World Health Organization recommends artemisinin-based combination therapies for the treatment of uncomplicated Plasmodium falciparum infections. However, the emergence of multidrug resistant strains of Plasmodium has necessitated the identification of new biological targets as well as the development of antimalarial chemotherapeutics with novel modes of action. Despite intensive research towards the identification of novel antimalarial chemotherapeutics that can kill the parasite, there remains a significant knowledge gap regarding how antimalarial therapeutics affect the biology of P. falciparum parasites. One functional genomics approach that can be used to identify the effects of drug perturbation on the biology of the parasite, is phenomics. Phenomics is defined as the collective characterization and quantification of the various phenotypes of an organism that describes the measurable physical and chemical interactions between genes and the environment and is expressed by either a cell, tissue or an organism. It aims to assess the global phenotypes in a set space and time under different environmental conditions. Phenomics provides an assessment of the biological pathways that are perturbed due to environmental conditions. In this dissertation, the phenome of P. falciparum parasites was evaluated in situ in response to antimalarial compounds as environmental perturbations. Specifically, the phenome was determined by using Phenotype MicroArray (PM) technology to measure the real-time ability of P. falciparum parasites to use a variety of carbohydrate substrates for energy production. This was extended to evaluate parasites perturbed by antimalarial drug treatment including classically used drugs such as chloroquine (CQ) disulphate, pyrimethamine (PYR) and dihydroartemisinin (DHA). Substrate catabolism was used to identify global differences between treated and untreated trophozoite-stage P. falciparum parasites. The information obtained was subsequently used to interrogate the presence of distinct phenotypic fingerprints consistent with the mode of action of each compound. Significant variations in carbohydrate source utilization were identified with, untreated parasites catabolizing 56% of the carbohydrate substrates. By contrast, parasites treated with the antimalarials were metabolically severely affected as evident in a use of only 13% to maximally 40% of the substrates. Global analyses of the resultant phenomes revealed clear differences in the phenotypic profiles and modes of actions (MOAs) of parasites treated with either CQ disulphate, DHA or PYR. This study describes the first real-time phenotypic profile of treated and untreated trophozoite-stage P. falciparum parasites. The PM platform has displayed its utility in the identification of phenotypic profiles and distinguishing between variations in MOAs of classical antimalarial compounds. This study serves as a blueprint for future PM studies of drug perturbations in the asexual form of the parasite.