Epigenetic regulation of virulence gene expression in plasmodium falciparum

Establishment and maintenance of infection by a pathogen relies on its ability to survive and grow in diverse host environments as well as successfully evade mounting immune responses. The human malaria parasite Plasmodium falciparum affects millions of people and causes over one million deaths annually. During its erythrocytic life cycle, which is associated with all of the symptoms of clinical malaria, the parasite must be able to both interact with the polymorphic surface of the red blood cell to initiate invasion and avoid immune clearance by the spleen by binding to host endothelial cells. Although these processes are mediated by distinct groups of proteins, many of these proteins share the common properties of being encoded by multigene families, which are highly polymorphic and often variantly expressed. Epigenetic mechanisms have been demonstrated to have a role in the regulation of the mutually exclusive expression of the var gene family, which is involved in cytoadherence, as the functions of two class III histone deacetylases are needed to maintain this tight regulation. Additionally, specific histone methylation marks have previously been associated with active and silenced var gene states. Here, we demonstrate the multigene RhopH1/clag subunit of the rhoptry body RhopH complex is variantly expressed in both field isolates and laboratory strains, and the silencing of at least two of its members is heterochromatin-mediated. Genetic experiments leading to the silencing of these two RhopH1/clag family members reveal that their expression is necessary for optimal parasite growth, providing the first genetic evidence of a functional role of this complex. Although examining histone modifications associated with specific gene expression states provides insight as to whether a gene is epigenetically regulated, understanding the epigenetic mechanism mediating these expression states requires the functional analysis of chromatin proteins that write, remove and read histone modifications. Here, we genetically disrupt a P. falciparum homolog to lysine-specific demethylase-1 and demonstrate a role for it in the silencing and temporal regulation of the var gene family. This is only the third chromatin modifying enzyme functionally characterized in Plasmodium spp. , and similar to Pf Sir2 A, PfLSD1 appears important in maintenance of mutually exclusive var gene silencing as well as telomere repeat length. This work provides the first functional evidence, to our knowledge, for a role of histone demethylase enzymes in controlling variant expression of virulence genes, and adds another layer to the complexity underlying the tight regulation of the var gene family. Attempts to phenocopy the PfSir2A and PfLSD1 knock-out parasites by treatment of wild-type parasites with known Sir2 and LSD1 inhibitors was unsuccessful, most likely due to the fact these enzymes are highly divergent in P. falciparum . Small molecule screens with libraries of related compounds may identify hits with high specificity against these enzymes, or better able to gain access into the parasite nucleus. Future studies should be aimed at further uncovering gene families which are epigenetically regulated as well as genetic and biochemical studies to determine the mechanism of this silencing. A better understanding of the processes mediating the tightly regulated expression of these genes is needed in order to devise strategies to successfully interfere with these virulence programs.