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