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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - SECOMAP (Sexual Commitment of Malaria Parasites: Investigation Into the Epigenetic Control of Plasmodium Gametocytogenesis)

Teaser

Summary

Despite major efforts to improve prevention and boost treatment malaria is still a major burden for global health. In 2015 the WHO recorded 212 million new cases and 429,000 deaths (http://www.who.int/malaria/publications/world-malaria-report-2016/report/en/). Plasmodium parasites, the causative agent of malaria, alternate between their mammalian host and their mosquito vector. In order to be transmitted to mosquitoes, malaria parasites must develop into gametocytes, which is the only Plasmodium form which can be taken up by mosquitoes in order to initiate a new cycle of infection. In order to maintain the current infection only a small number of parasites circulating in the hostâ€™s blood stream undergo gametocytogenesis and develop into gametocytes. Transmission from host to mosquito is considered as a bottle neck in the parasite life cycle, representing a possible target for pharmacological intervention which would disrupt the spread of malaria. However, only little is known about the molecular mechanisms during gametocytogenesis and only recently its master regulator, the transcription factor AP2-G, was identified. AP2-G is believed to be epigenetically silenced in the majority of circulating malaria parasites and only (stochastically) activated in a small number of cells, triggering commitment to gametocytogenesis. The objective of this project was to identify and characterize possible candidate enzymes involved in the epigenetic regulation of Plasmodium development in general, and of ap2-g in particular.

Work performed

In order to identify proteins involved in the epigenetic regulation of AP2-G, we first focused on candidates predicted to catalyse the methylation or demethylation of histones. The modification of histone tails with methyl groups (e.g. H3K9me3) is known to be a key epigenetic mechanism regulating gene activation and repression in eukaryotic cells, and is believed to be involved in the silencing of AP2-G. In the first part of the project, we generated Plasmodium berghei (a model malaria species infectious to rodents) single knock-out (KO) lines deficient for any of the nuclear localized histone lysine methyl transferase (HKMTs, 8 identified) or histone lysine demethylase (HKDMs, 2 identified) encoded in the P. berghei genome. We successfully generated 6 P. berghei HKMT-KO lines and 2 HKDM-KO lines, but failed repeatedly to delete the remaining 4 HKMTs. All successfully generated KO lines were then further phenotypically screened for effects on growth and gametocyte level in blood stage parasites, as well as for possible defects during development within the mosquito vector. This allowed us to identify a HKMT-KO line displaying a markedly reduced growth rate as well as an increased number of gametocytes. In ongoing work we attempt to further characterize the identified HKMT and its role in Plasmodium life-cycle progression and to verify its importance during gametocytogenesis.
The repeated failure to generate KO mutants of the remaining 4 HKMTs lead us to assume that those proteins are most likely essential for haploid parasite development, and hence their deletion was lethal for the parasite. To investigate the role of those essential HKMTs, we employed a conditional KO (cKO) approach, based on the Auxin Inducible Degradation (AID) system. This system allows the conditional degradation of proteins tagged with and AID-degron upon the addition of the plant hormone Auxin. In order to employ the AID system, we generated P. berghei parent lines (expressing the plant Auxin receptor protein TIR1) which would allow the degradation of AID-tagged proteins as well as to monitor gametocyte development (generating GFP expressing male and RFP expressing female gametocytes). Subsequently, we successfully tagged all identified HKMTs and HKDMs with an AID degron in the background of the AID parent line. Unfortunately, upon addition of Auxin we observed limited degradation efficiency, most likely due to the nuclear localisation of the target proteins. Hence, ongoing work currently focuses on the improvement and adaptation of the AID cKO system in order to allow sufficient degradation of nuclear proteins and their subsequent characterisation.

The results of the MSCA were disseminated through presentations at the following international conferences:

â€¢ MAM 2016 â€“ Molecular Approaches to Malaria, 21-25 February 2016, Lorne, Australia
â€¢ BioMalPar XII: Biology and Pathology of the Malaria Parasite, 18-20 May 2016, Heidelberg, Germany
â€¢ BSP Spring Meeting 2017, 2-5 April 2016, Dundee, United Kingdom
â€¢ BioMalPar XIII: Biology and Pathology of the Malaria Parasite, 29-31 May 2017, Heidelberg, Germany

Final results

Gene expression regulation through the modification of histones is a key aspect of Plasmodium biology. Among the many modifications, methylation of histone tails is one of the main epigenetic regulatory mechanisms in eukaryotic cells and has also been demonstrated to play a role in Plasmodium gene expression regulation. In particular, the H3K9me3 histone mark has been proposed to repress the expression of the transcription factor AP2-G, which is the master regulator of gametocytogenesis. However, only few of the key players responsible for methylation or demethylation of histones in Plasmodium have been characterised comprehensively, and no histone writer silencing the AP2-G locus has been identified so far. To our knowledge, this project is the first in depth investigation of the role in gametocytogenesis of all identified HKMTs and HKDMs in malaria parasites. The usage of the rodent infectious malaria parasite P. berghei allowed us not only to investigate the role of HKMTs and HKDMs during the intraerythrocytic development cycle, but also during mosquito development. So far, none of the methyl transferases or demethylases has been investigated with respect to their role during the development within the mosquito vector. Ultimately, the results obtained during this MSCA set the framework for further detailed investigations into the role of HKMTs and HKDMs in Plasmodium development using traditional KO as well as inducible cKO approaches. In particular the usage of the improved AID-based cKO system will strongly facilitate the investigations of the histone modifier essential for Plasmodium survival and hence refractory to direct KO approaches. In addition the new approaches should prove useful for the study of other nuclear processes in Plasmodium (3D organisation, transcription, splicing). Overall, malaria can have devastating effects on human health; it is a major burden for global health and a main driver of poverty with a significant knock on effect on the global economy. Combating malaria is one of the major health goals of the European Union and will also contribute to the wellbeing and prosperity of European society. The findings of this project have laid the groundwork for further investigations into the epigenetic mechanisms regulating Plasmodium development and will hopefully strongly facilitate our knowledge of the malaria parasite biology, with possible implications for therapeutic intervention in malaria infection and propagation.