Integrative transcriptome and phenome analysis reveals unique regulatory cascades controlling the intraerythrocytic asexual and sexual development of human malaria parasites

The Plasmodium falciparum parasite, the major causative agent of malaria on the African continent, has evolved numerous cellular adaptations to effectively propagate its species. The parasite can proliferate asexually, producing mass amounts of progeny to subsist in the human host or differentiate into sexual forms (gametocytes) that, once mature, can transmit to a feeding Anopheles mosquito. Key to our ability to effectively develop chemical candidates that interfere with either of these processes is the identification and understanding of critical factors that regulate parasite development. This is particularly true for the development of antimalarials that can be used in malaria elimination strategies by targeting both parasite proliferation and transmission. We therefore hypothesized that parasite proliferation and differentiation use divergent mechanisms for gene expression that could be observed through a thorough investigation of the functional genome of these different parasite forms. This doctoral study therefore set out to increase our knowledge base on three crucial aspects of parasite development: 1) the atypical cell cycle that allows the rapid proliferation of asexual parasites; 2) the full molecular profile of gametocytogenesis enabling the cellular differentiation that allows the parasite to transmit; and 3) the metabolic differences between these proliferating and differentiating parasites that results from their strategy-specific mechanisms of developmental control. The atypical cell cycle of the parasite, associated with the massive cell number expansion in asexual development, is notoriously difficult to study. Here, we contributed a novel system by developing a cell cycle synchronization tool that reversibly blocks the development of asexual parasites at the G1/S transition. This results in an inescapable arrest of the cell cycle that is completely and functionally reversible; parasites re-initiate cell cycle progression and continue to S phase within 6 h. This system provided the opportunity to characterize cell cycle phases in the parasite and additionally evaluate molecular mechanisms associated with cell cycle arrest or re-initiation. During cell cycle arrest, the parasite enters a quiescent state reminiscent of a mitogen-activated restriction point. This arrest is unique and solely attributed to the removal of the specific mitogens within this system, polyamines. These analyses indicate the close interaction between transcriptional regulation and signal transduction cascades in the progression through the parasite’s cell cycle and for the first time highlight aspects of controlled cell cycle regulation in Plasmodium. In contrast to proliferation, the process of sexual differentiation only started receiving attention in the past few years. As such, we lack fundamental understanding of the mechanisms driving the unique gametocyte differentiation of P. falciparum parasites. This study contributes a detailed analysis of gametocyte differentiation that revealed distinct developmental transitions demarcating the start of gametocytogenesis, intermediate gametocyte development and finally maturation to produce the transmissible mature gametocytes. The study provides evidence for coordinated regulation of gene expression on a transcriptional level. We propose a model for regulation of gametocytogenesis in malaria parasites that involves active repression of gene sets mediated through epigenetics and RNA destabilization as well as active transcription of gene sets through successive ApiAP2 transcription factor activity. This data provides the most detailed framework of coordinated gene regulation events underlying development of P. falciparum gametocytes to date, a unique resource for the malaria community. The comprehensive and complex transcriptional regulation described for the proliferation and differentiation of the parasite led us to evaluate the functional consequence thereof. A whole cell phenotype microarray system was evaluated for its ability to measure the metabolic processes that define asexual and sexual stage metabolism as a functional consequence of changed gene expression profiles during proliferation and differentiation. The study provided metabolic profiles detailing carbon and nitrogen metabolism in asexual parasites, mature and immature gametocyte stages. The data highlighted dipeptide metabolism as a distinguishing feature in mature gametocytes and showed the presence of a low, delayed metabolic state concurrent with reduced transcriptional activity observed in this stage. These results show that gene expression changes associated with differentiation compared to proliferation translate to an observable metabolic phenotype and that transcriptional regulation shapes the molecular landscape underlying crucial events that enable the parasite’s intraerythrocytic asexual and sexual development.Thesis (PhD)--University of Pretoria, 2019.BiochemistryPhDUnrestricte

Quantitative chromatin proteomics reveals a dynamic histone posttranslational modification landscape that defines asexual and sexual Plasmodium falciparum parasites

Gene expression in Plasmodia integrates post-transcriptional 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 entire asexual and sexual developmental cycles of the parasite, we used complementary and comparative quantitative chromatin proteomics to identify and functionally characterise histone PTMs in 8 distinct life cycle stages of P. falciparum parasites. ~500 individual histone PTMs were identified of which 106 could be stringently validated. 46 individual histone PTMs and 30 co-existing PTMs were fully quantified with high confidence. Importantly, 15 of these histone PTMs are novel for Plasmodia (e.g. H3K122ac, H3K27me3, H3K56me3). The comparative nature of the data revealed a highly dynamic histone PTM landscape during life cycle development, with a set of histone PTMs (H3K4ac, H3K9me1 and H3K36me2) displaying a unique and conserved abundance profile exclusively during gametocytogenesis (P < 0.001). Euchromatic histone PTMs are abundant during schizogony and late gametocytes; heterochromatic PTMs mark early gametocytes. Collectively, this data provides the most accurate, complete and comparative chromatin proteomic analyses of the entire life cycle development of malaria parasites. A substantial association between histone PTMs and stage-specific transition provides insights into the intricacies characterising Plasmodial developmental biology.The South African National Research Foundation (FA2007050300003 & UID 84627), the Medical Research Council and the European Community’s Seventh Framework Programme (FP7/2007–2013, No. 242095) to LB and a PhD Innovation Bursary from the NRF to N.C. B.G. received funding for this work from the US NIH (R01 GM110174 and AI118891).http://www.nature.com/scientificreportsam2017Biochemistr

Interrogating alkyl and arylalkylpolyamino (bis)urea and (bis)thiourea isosteres as potent antimalarial chemotypes against multiple lifecycle forms of Plasmodium falciparum parasites

A new series of potent potent aryl/alkylated (bis)urea- and (bis)thiourea polyamine analogues were synthesized and evaluated in vitro for their antiplasmodial activity. Altering the carbon backbone and terminal substituents increased the potency of analogues in the compound library 3-fold, with the most active compounds, 15 and 16, showing half-maximal inhibitory concentrations (IC50 values) of 28 and 30 nM, respectively, against various Plasmodium falciparum parasite strains without any cross-resistance. In vitro evaluation of the cytotoxicity of these analogues revealed marked selectivity towards targeting malaria parasites compared to mammalian HepG2 cells (>5000-fold lower IC50 against the parasite). Preliminary biological evaluation of the polyamine analogue antiplasmodial phenotype revealed that (bis)urea compounds target parasite asexual proliferation, whereas (bis)thiourea compounds of the same series have the unique ability to block transmissible gametocyte forms of the parasite, indicating pluripharmacology against proliferative and non-proliferative forms of the parasite. In this manuscript, we describe these results and postulate a refined structure–activity relationship (SAR) model for antiplasmodial polyamine analogues. The terminally aryl/alkylated (bis)urea- and (bis)thiourea–polyamine analogues featuring a 3-5-3 or 3-6-3 carbon backbone represent a structurally novel and distinct class of potential antiplasmodials with activities in the low nanomolar range, and high selectivity against various lifecycle forms of P. falciparum parasites.South African National Research Foundation (FA2007050300003 & UID: 84627), the University of Pretoria and the South African Medical Research Council Strategic Health Initiatives Partnerships with the Medicines for Malaria Venture.http://www.elsevier.com/locate/bmc2016-08-31hb201

Potent Plasmodium falciparum gametocytocidal compounds identified by exploring the kinase inhibitor chemical space for dual active antimalarials

OBJECTIVES : Novel chemical tools to eliminate malaria should ideally target both the asexual parasites and transmissible gametocytes. Several imidazopyridazines (IMPs) and 2-aminopyridines (2-APs) have been described as potent antimalarial candidates targeting lipid kinases. However, these have not been extensively explored for stage-specific inhibition of gametocytes in Plasmodium falciparum parasites. Here we provide an in-depth evaluation of the gametocytocidal activity of compounds from these chemotypes and identify novel starting points for dual-acting antimalarials. METHODS : We evaluated compounds against P. falciparum gametocytes using several assay platforms for cross-validation and stringently identified hits that were further profiled for stage specificity, speed of action and ex vivo efficacy. Physicochemical feature extraction and chemogenomic fingerprinting were applied to explore the kinase inhibition susceptibility profile. RESULTS : We identified 34 compounds with submicromolar activity against late stage gametocytes, validated across several assay platforms. Of these, 12 were potent at 1000-fold selectivity towards the parasite over mammalian cells. Front-runner compounds targeted mature gametocytes within 48 h and blocked transmission to mosquitoes. The resultant chemogenomic fingerprint of parasites treated with the lead compounds revealed the importance of targeting kinases in asexual parasites and gametocytes. CONCLUSIONS : This study encompasses an in-depth evaluation of the kinase inhibitor space for gametocytocidal activity. Potent lead compounds have enticing dual activities and highlight the importance of targeting the kinase superfamily in malaria elimination strategies.The South African Medical Research Council (SAMRC) Self-initiated Research (to JN) and Strategic Health Initiatives Partnerships (MRC-SHIP) programmes to L.B., T.C., D.M. K.C. further acknowledges the SAMRC for funding of the extramural Drug Discovery and Development Research Unit at UCT. The SAMRC is acknowledged for funding of the UP ISMC (LMB) and WRIM (TLC) as Collaborating Centres for Malaria Research. The Council for Scientific and Industrial Research and the 3R Foundation (project 118–10) to D.M. We thank the Medicines for Malaria Venture and South African Technology Innovation Agency (TIA) for funding to K.C. (Project MMV09/0002). The University of Cape Town, University of Pretoria, and South African Research Chairs Initiative of the Department of Science and Technology, administered through the South African National Research Foundation are gratefully acknowledged for support to K.C. and L.B. (UID84627). JN was supported through an International Society for Infectious Diseases grant.https://academic.oup.com/jac2019-05-01hj2018Biochemistr

Hierarchical transcriptional control regulates Plasmodium falciparum sexual differentiation

BACKGROUND : Malaria pathogenesis relies on sexual gametocyte forms of the malaria parasite to be transmitted between the infected human and the mosquito host but the molecular mechanisms controlling gametocytogenesis remains poorly understood. Here we provide a high-resolution transcriptome of Plasmodium falciparum as it commits to and develops through gametocytogenesis. RESULTS : The gametocyte-associated transcriptome is significantly different from that of the asexual parasites, with dynamic gene expression shifts characterizing early, intermediate and late-stage gametocyte development and results in differential timing for sex-specific transcripts. The transcriptional dynamics suggest strict transcriptional control during gametocytogenesis in P. falciparum, which we propose is mediated by putative regulators including epigenetic mechanisms (driving active repression of proliferation-associated processes) and a cascade-like expression of ApiAP2 transcription factors. CONCLUSIONS : The gametocyte transcriptome serves as the blueprint for sexual differentiation and will be a rich resource for future functional studies on this critical stage of Plasmodium development, as the intraerythrocytic transcriptome has been for our understanding of the asexual cycle.Additional File 1: Table S1 Total microarray data with GO enrichment pertaining to Fig. 1 & 2Additional File 2. Correlation of microarray time points and gametocyte markers pertaining to Fig. 1Additional File 3. Cross-dataset comparison and functional enrichment pertaining to Figs. 2-5Additional file 4: Fig. S1. qPCR validation of select gametocyte genes. Fig. S2. Transcript abundance of ApiAP2 transcription factors during P. falciparum gametocyte development. Fig. S3. Transcript abundance of ap2-g and downstream genes (identified in Josling et al. 2019)The South African Medical Research Council and the South African Research Chairs Initiative of the Department of Science and Technology, administered through the South African National Research Foundation (UID 84627) and the European Commission ‘EviMalar” (no 242095) to LMB.https://bmcgenomics.biomedcentral.comhj2020BiochemistryGeneticsMicrobiology and Plant Patholog

Chemogenomic fingerprints associated with stage-specific gametocytocidal compound action against human malaria parasites

Please read abstract in the article.Supplementary material 1: Figure S1: MMV390048 or MMV642943 chemical structures. Figure S2: Verification of microarray-identified differential gene expression with qPCR analyses (PDF)Supplementary material 2: Additional Excel File provided with a summary of the full gene expression data set with GO and cluster analyses (XLSX)The South African Medical Research Council and the Department of Science and Innovation South African Research Chairs Initiative Grants managed by the National Research Foundation and the International Society of Infectious diseases Small Grants program.https://pubs.acs.org/journal/aidcbchj2021BiochemistryGeneticsMicrobiology and Plant PathologyUP Centre for Sustainable Malaria Control (UP CSMC