Sexual differentiation of malaria parasites is controlled by unique epigenomic and proteomic cascades as revealed by comparative functional genome analyses

Abstract

There is a continuous and urgent need for novel antimalarial agents with new modes of action due to P. falciparum resistance development against most of the currently used therapeutics. In the search for a novel class of antimalarial, the identification of a novel target, preferably present in both the asexual and sexual gametocyte stages, is essential. P. falciparum gametocytes develop due to the irreversible binary choice of a minor proportion of asexual parasites to commit to sexual differentiation. This process is characterised by essential morphological, physiological and biochemical changes to prepare the terminally differentiated mature gametocytes for transmission to the mosquito. It is therefore undeniably important to target these transmissible stages using chemotherapeutic interventions that, in context of limiting resistance development, should be exploited by means of novel drug targets. However, the complexity of gametocytogenesis and the stage-specific developmental decisions is not fully understood and this impedes the discovery of essential molecular players that can be targeted in intervention strategies. In this project, stage-specific gametocytogenesis was studied at the level of epigenome regulation through quantitative chromatin proteomics, mirrored by evaluation of the dynamics in the global quantitative proteome during this differentiation process. Ultimately, the information obtained with these global evaluation strategies informed analyses of novel inhibitors that target the parasite’s epigenetic gene regulation machinery. Gene expression in Plasmodia integrates post-transcriptional and translational regulation with epigenetic marking of active genomic regions through histone post-translational modifications (PTMs). To generate insights into the importance of histone PTMs to the parasite’s entire asexual and sexual developmental cycles, we used comparative quantitative chromatin proteomics to identify and functionally characterise eight distinct P. falciparum life cycle stages. Various novel histone PTMs and stage-specific histone PTM profiles were identified, with histone PTM combinations classified for the first time in P. falciparum. Gametocytes were enriched for a specific set of histone PTMs that is involved in stage-specific differentiation. This stage-specific differentiation was subsequently also observed in a global comparative quantitative proteome evaluation of gametocytogenesis. Protein abundance profiles and functional annotation of protein sets led to the description of enriched biological processes during stage-specific gametocyte development. Interestingly, gametocyte sex differentiation was shown to be characterised by key molecular players very early on during development, and genes previously identified as translationally repressed were shown here to be directly involved in gametocyte development. The importance of chromatin level regulation and its observed effect on the proteome dynamics during gametocytogenesis led to the proposal that these essential processes could be targeted with intervention strategies. We discovered compounds with limited cross-resistance and potent multi-stage activity against asexual parasites, early and late stage gametocytes by targeting the parasite’s epigenetic regulation. Collectively, this thesis presents the most complete and comparative chromatin proteomic and global proteomic analyses of the entire stage-specific P. falciparum development, providing insights into the intricacies characterising Plasmodial developmental biology, specifically during gametocytogenesis. The data importantly prove that novel epigenetic regulatory mechanisms identified in this study can be targeted with chemotherapeutic interventions, overcoming current resistance mechanisms and contribute to malaria elimination strategies.