Genetic manipulation of histone deacetylases SAP18 and SIR2A in Plasmodium falciparum

Abstract

Malaria parasites are increasingly developing resistance to current drug treatments, emphasizing the continued need for new drugs targeting the disease-causing parasite, Plasmodium falciparum and the investigation of targetable biological processes. This includes the unique and unusual epigenome of the parasite, where epigenetic mechanisms regulate both asexual proliferation and sexual differentiation. Here, we investigate the putative histone deacetylase I complex protein, PF3D7_0711400 (SAP18), and a member of the histone deacetylase III family, PF3D7_1328800 (SIR2A). These proteins are thought to regulate histone acetylation, thereby allowing repression of genes in a very specific manner. These genes also show a unique expression profile during cell cycle arrest and re-entry. This phenomenon indicates an important role of both SAP18 and SIR2A in cell cycle progression. This study aimed to manipulate sap18 and sir2A for the investigation of essentiality and localisation in P. falciparum parasites. Sap18 and sir2A were targeted for gene disruption to prove their functional essentiality. The DNA sequence encoding the N-terminus of sap18 and sir2A was manipulated into a specialised gene disruption plasmid (pSLI-TGD) that allows antibiotic-driven selection of integration of truncated sap18 and sir2A genes into the parasite genome. Additionally, these genes were tagged with green fluorescent protein (GFP) to allow for localisation of the protein during asexual proliferation by manipulating the C-terminus of sap18 and sir2A. The pSLI-Sandwich/pLyn-FRB-nmd3-BSD-mCherry plasmids, also allowed for mislocalisation of the protein of interest. This was used to achieve knock sideways of SAP18, however, no effect on asexual proliferation was observed. GFP fluorescence showed that SAP18 localises to the nucleus. Disruption of sap18 using pSLI-TGD does not produce a growth defect, further supporting the conclusion that SAP18 is not essential in asexual stages. The disruption of SAP18 was evaluated for its effect in cell cycle arrest and re-entry. These parasites arrest in early trophozoite stage and re-enter the cell cycle after the addition of putrescine in the same manner as wild type parasites. These data suggest that SAP18 may not be a major regulator of HDAC1 activity, which differentiates P. falciparum from other organisms. This study has allowed for evaluation of the functional importance of SAP18 to parasite survival and has the potential to provide novel tools for analysis of the epigenetic regulation of the cell cycle in P. falciparum parasites.