Histone Modification Landscapes as a Roadmap for Malaria Parasite Development.

Plasmodium falciparum remains the deadliest parasite species in the world, responsible for 229 million cases of human malaria in 2019. The ability of the P. falciparum parasite to progress through multiple life cycle stages and thrive in diverse host and vector species hinges on sophisticated mechanisms of epigenetic regulation of gene expression. Emerging evidence indicates such epigenetic control exists in concentric layers, revolving around core histone post-translational modification (PTM) landscapes. Here, we provide a necessary update of recent epigenome research in malaria parasites, focusing specifically on the ability of dynamic histone PTM landscapes to orchestrate the divergent development and differentiation pathways in P. falciparum parasites. In addition to individual histone PTMs, we discuss recent findings that imply functional importance for combinatorial PTMs in P. falciparum parasites, representing an operational histone code. Finally, this review highlights the remaining gaps and provides strategies to address these to obtain a more thorough understanding of the histone modification landscapes that are at the center of epigenetic regulation in human malaria parasites.

In vitro inhibition of Plasmodium falciparum early and late stage gametocyte viability by extracts from eight traditionally used South African plant species.

ETHNOPHARMACOLOGICAL RELEVANCE: Extracts of plant species, used traditionally to treat malaria, have been extensively investigated for their activity against Plasmodium intraerythrocytic asexual parasites in search of new antimalarial drugs. However, less effort has been directed towards examining their efficacy in blocking transmission. Here, we report the results of the in vitro screening of extracts from eight selected plant species used traditionally to treat malaria in South Africa for activity against Plasmodium falciparum NF54 early and late stage gametocytes. The species used were Khaya anthotheca, Trichilia emetica, Turraea floribunda, Leonotis leonurus, Leonotis leonurus ex Hort, Olea europaea subsp. Africana, Catha edulis and Artemisia afra. AIM OF THE STUDY: To investigate the activities of extracts from plant species traditionally used for malaria treatment against P. falciparum gametocytes. MATERIAL AND METHODS: Air-dried and ground plant leaves were extracted using acetone. Primary two point in vitro phenotypic screens against both early and late stage gametocytes were done at 10 and 20µg/ml followed by full IC50 determination of the most active extracts. Inhibition of gametocyte viability in vitro was assessed using the parasite lactate dehydrogenase (pLDH) assay. RESULTS: Of the eight crude acetone extracts from plant species screened in vitro, four had good activity with over 50-70% inhibition of early and late stage gametocytes' viability at 10 and 20µg/ml, respectively. Artemisia afra (Asteraceae), Trichilia emetica (Meliaceae) and Turraea floribunda (Meliaceae) were additionally highly active against both gametocyte stages with IC50 values of less than 10µg/ml while Leonotis leonurus ex Hort (Lamiaceae) was moderately active (IC50<20µg/ml). The activity of these three highly active plant species was significantly more pronounced on late stage gametocytes compared to early stages. CONCLUSION: This study shows the potential transmission blocking activity of extracts from selected South African medicinal plants and substantiates their traditional use in malaria control that broadly encompasses prevention, treatment and transmission blocking. Further studies are needed to isolate and identify the active principles from the crude extracts of A. afra, T. emetica and T. floribunda, as well as to examine their efficacy towards blocking parasite transmission to mosquitoes.

Polyamine uptake by the intraerythrocytic malaria parasite, Plasmodium falciparum.

Polyamines and the enzymes involved in their biosynthesis are present at high levels in rapidly proliferating cells, including cancer cells and protozoan parasites. Inhibition of polyamine biosynthesis in asexual blood-stage malaria parasites causes cytostatic arrest of parasite development under in vitro conditions, but does not cure infections in vivo. This may be due to replenishment of the parasite's intracellular polyamine pool via salvage of exogenous polyamines from the host. However, the mechanism(s) of polyamine uptake by the intraerythrocytic parasite are not well understood. In this study, the uptake of the polyamines, putrescine and spermidine, into Plasmodium falciparum parasites functionally isolated from their host erythrocyte was investigated using radioisotope flux techniques. Both putrescine and spermidine were taken up into isolated parasites via a temperature-dependent process that showed cross-competition between different polyamines. There was also some inhibition of polyamine uptake by basic amino acids. Inhibition of polyamine biosynthesis led to an increase in the total amount of putrescine and spermidine taken up from the extracellular medium. The uptake of putrescine and spermidine by isolated parasites was independent of extracellular Na(+) but increased with increasing external pH. Uptake also showed a marked dependence on the parasite's membrane potential, decreasing with membrane depolarization and increasing with membrane hyperpolarization. The data are consistent with polyamines being taken up into the parasite via an electrogenic uptake process, energised by the parasite's inwardly negative membrane potential.

Functional consequences of perturbing polyamine metabolism in the malaria parasite, Plasmodium falciparum.

Inhibition of polyamine biosynthesis and/or the perturbation of polyamine functionality have been exploited with success against parasitic diseases such as Trypanosoma infections. However, when the classical polyamine biosynthesis inhibitor, alpha-difluoromethylornithine, is used against the human malaria parasite, Plasmodium falciparum, it results in only a cytostatic growth arrest. Polyamine metabolism in this parasite has unique properties not shared by any other organism. These include the bifunctional arrangement of the catalytic decarboxylases and an apparent absence of the typical polyamine interconversion pathways implying different mechanisms for the regulation of polyamine homeostasis that includes the uptake of exogenous polyamines at least in vitro. These properties make polyamine metabolism an enticing drug target in P. falciparum provided that the physiological and functional consequences of polyamine metabolism perturbation are understood. This review highlights our current understanding of the biological consequences of inhibition of the biosynthetic enzymes in the polyamine pathway in P. falciparum as revealed by several global analytical approaches. Ultimately, the evidence suggests that polyamine metabolism in P. falciparum is a validated drug target worth exploiting.

Antimalarial drug discovery: in silico structural biology and rational drug design.

Malaria remains one of the most burdensome human infectious diseases, with a high rate of resistance outbreaks and a constant need for the discovery of novel antimalarials and drug targets. For several reasons, Plasmodial proteins are difficult to characterise structurally using traditional physical approaches. However, these problems can be partially overcome using a number of in silico approaches. This review describes the peculiarities of malaria proteins and then details various in silico strategies to select and allow descriptions of the molecular structures of drug target candidates as well as subsequent rational approaches for drug design. Chiefly, homology modelling with specific focus on unique aspects of malaria proteins including low homology, large protein size and the presence of parasite-specific inserts is addressed and alternative strategies including multiple sequence and structure-based prediction methods, sampling-based approaches that aim to reveal likely global or shared features of a Plasmodial structure and the value of molecular dynamics understanding of unique features of Plasmodial proteins are discussed. Once a detailed description of the drug target is available, in silico approaches to the specific design of an inhibitory drug thereof becomes invaluable as an economic and rational alternative to chemical library screening.

Exploring functional genomics for drug target and therapeutics discovery in Plasmodia.

Functional genomics approaches are indispensable tools in the drug discovery arena and have recently attained increased attention in antibacterial drug discovery research. However, the application of functional genomics to post-genomics research of Plasmodia is still in comparatively early stages. Nonetheless, with this genus having the most species sequenced of any eukaryotic organism so far, the Plasmodia could provide unique opportunities for the study of intracellular eukaryotic pathogens. This review presents the status quo of functional genomics of the malaria parasite including descriptions of the transcriptome, proteome and interactome. We provide examples for the in silico mining of the X-ome data sets and illustrate how X-omic data from drug challenged parasites might be used in elucidating amongst others, the mode-of-action of inhibitory compounds, validate potential targets and discover novel targets/therapeutics.

Comparative properties of a three-dimensional model of Plasmodium falciparum ornithine decarboxylase.

The ornithine decarboxylase (ODC) component of the bifunctional S-adenosylmethionine decarboxylase/ornithine decarboxylase enzyme (PfAdoMetDC-ODC) of Plasmodium falciparum was modeled on the crystal structure of the Trypanosoma brucei enzyme. The homology model predicts a doughnut-shaped active homodimer that associates in a head-to-tail manner. The monomers contain two distinct domains, an N-terminal alpha/beta-barrel and a C-terminal modified Greek-key domain. These domains are structurally conserved between eukaryotic ODC enzymes and are preserved in distant analogs such as alanine racemase and triosephosphate isomerase-like proteins. Superimposition of the PfODC model on the crystal structure of the human enzyme indicates a significant degree of deviation in the carbon alpha-backbone of the solvent accessible loops. The surface locality of the ab initio modeled 38 amino acid parasite-specific insert suggests a role in the stabilization of the large bifunctional protein complex. The active site pockets of PfODC at the interface between the monomers appear to be conserved regarding the binding sites of the cofactor and substrate, but each contains five additional malaria-specific residues. The predicted PfODC homology model is consistent with mutagenesis results and biochemical studies concerning the active site residues and areas involved in stabilizing the dimeric form of the protein. Two competitive inhibitors of PfODC could be shown to interact with several parasite-specific residues in comparison with their interaction with the human ODC. The PfODC homology model contributes toward a structure-based approach for the design of novel malaria-specific inhibitors.