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Pregnancy-associated malaria commonly involves the binding of Plasmodium falciparum-infected erythrocytes to placental chondroitin sulfate A (CSA) through the PfEMP1-VAR2CSA protein. VAR2CSA is translationally repressed by an upstream open reading frame. In this study, we report that the P. falciparum translation enhancing factor (PTEF) relieves upstream open reading frame repression and thereby facilitates VAR2CSA translation. VAR2CSA protein levels in var2csa-transcribing parasites are dependent on the expression level of PTEF, and the alleviation of upstream open reading frame repression requires the proteolytic processing of PTEF by PfCalpain. Cleavage generates a C-terminal domain that contains a sterile-alpha-motif-like domain. The C-terminal domain is permissive to cytoplasmic shuttling and interacts with ribosomes to facilitate translational derepression of the var2csa coding sequence. It also enhances translation in a heterologous translation system and thus represents the first non-canonical translation enhancing factor to be found in a protozoan. Our results implicate PTEF in regulating placental CSA binding of infected erythrocytes.

Background: Protein kinase CK2 is a pleiotropic serine/threonine protein kinase with hundreds of reported substrates, and plays an important role in a number of cellular processes. The cellular functions of Plasmodium falciparum CK2 (PfCK2) are unknown. The parasite's genome encodes one catalytic subunit, PfCK2α, which we have previously shown to be essential for completion of the asexual erythrocytic cycle, and two putative regulatory subunits, PfCK2β1 and PfCK2β2., Results: We now show that the genes encoding both regulatory PfCK2 subunits (PfCK2β1 and PfCK2β2) cannot be disrupted. Using immunofluorescence and electron microscopy, we examined the intra-erythrocytic stages of transgenic parasite lines expressing hemagglutinin (HA)-tagged catalytic and regulatory subunits (HA-CK2α, HA-PfCK2β1 or HA-PfCK2β2), and localized all three subunits to both cytoplasmic and nuclear compartments of the parasite. The same transgenic parasite lines were used to purify PfCK2β1- and PfCK2β2-containing complexes, which were analyzed by mass spectrometry. The recovered proteins were unevenly distributed between various pathways, with a large proportion of components of the chromatin assembly pathway being present in both PfCK2β1 and PfCK2β2 precipitates, implicating PfCK2 in chromatin dynamics. We also found that chromatin-related substrates such as nucleosome assembly proteins (Naps), histones, and two members of the Alba family are phosphorylated by PfCK2α in vitro., Conclusions: Our reverse-genetics data show that each of the two regulatory PfCK2 subunits is required for completion of the asexual erythrocytic cycle. Our interactome study points to an implication of PfCK2 in many cellular pathways, with chromatin dynamics being identified as a major process regulated by PfCK2. This study paves the way for a kinome-wide interactomics-based approach to elucidate protein kinase function in malaria parasites.

Clonally variant gene families underlie phenotypic plasticity in Plasmodium falciparum, a process indispensable for survival of the pathogen in its human host. Differential transcription of one of these gene families in clonal parasite lineages has been associated with chromatin modifications. Here, we determine the genome-wide distribution in P. falciparum of a histone mark of heterochromatin, trimethylation of histone H3 lysine 9 (H3K9me3), using high-resolution ChIP-chip analysis. We show that H3K9me3 is specifically associated with clonally variant gene families, which are clustered on subtelomeric and some chromosome internal regions. High levels of H3K9me3 correlate with genes localized to the nuclear periphery, implying chromosome loop formation. Disruption of the histone deacetylase PfSir2 causes changes in H3K9me3 that are discontinuous along chromosomes and associated with disrupted monoallelic transcription. Our data point to the existence of perinuclear repressive centers associated with control of expression of malaria parasite genes involved in phenotypic variation and pathogenesis.

Protozoan pathogens have evolved countermeasures to avoid immune clearance and prolong the period of infection in their vertebrate hosts. The type and degree of immune escape strategies depends on the in vivo 'lifestyle' the pathogen has adopted. Here we describe how parasites use different strategies to coordinate their expression of phenotypic variation, which is used in many cases to fool the immune system, or to successfully invade new host cells. Recent insights using modern molecular biology techniques show that this is achieved via a coordinated manner of action of different epigenetic factors such as histone marks, subnuclear localization, or novel unknown mechanism(s). This emerging field may have an enormous impact on disease therapy.