Human malaria is caused by infection with intracellular protozoan parasites of the genus Plasmodium that are transmitted by Anopheles mosquitoes. Of four species that infect humans, Plasmodium falciparum is responsible for the most virulent form of the disease. The transition from one stage of the life cycle to the next must be tightly regulated, to ensure proliferation and differentiation occur when and where appropriate; this is undoubtedly linked to differential gene expression. Analysis of the P. falciparum transcriptome during the erythrocytic asexual cycle reveals an ordered cascade of gene expression , and the various developmental stages display distinct transcriptomes; how this is orchestrated remains obscure. Initial investigation of the P. falciparum genome revealed a paucity of transcriptional regulators , although this picture has recently been challenged by the recent identification of the ApiAP2 transcription factor family . There is nevertheless a large body of evidence suggesting that post-transcriptional control is an important means of gene regulation in P. falciparum. Examples include the relatively small number of identifiable transcription-associated proteins, abundance of CCCH-type zinc finger proteins commonly involved in modulating mRNA decay and translation rates  and translational repression during gametocytogenesis [4–6].
In mammalian cells, regulation of gene expression is a key mechanism in the mediation of stress responses, which may be achieved by influencing transcription or translation. The Stress Activated Protein kinases (SAPKs), specifically JNKs and p38 kinases, are subfamilies of mitogen activated protein kinases (MAPK) that are expressed in most eukaryotic cells, and respond to a variety of stress conditions . Although the parasite kinome includes two MAPK homologues, none of these are members of the SAPK subfamily [8–10]. In contrast, the P. falciparum kinome contains a phylogenetic cluster of three kinases with homology to eukaryotic Initiation Factor 2α (eIF2α kinases, which in other organisms regulate translation in response to stress . Interestingly, the related apicomplexan parasite Toxoplasma gondii has been shown to differentiate from tachyzoites to bradyzoites on exposure to a number of cellular stresses, concomitant with an increase in phosphorylation of TgeIF2α, indicating a possible role for this mechanism in parasite differentiation .
Phosphorylation of eukaryotic initiation factor 2α at residue Ser51 in response to stress is a well-characterized mechanism of post-transcriptional control that regulates initiation of translation [12–17]. In mammalian cells this phosphorylation event is mediated by four distinct protein kinases, called the eIF2α kinases: general control non-derepressible-2 (GCN2), haem-regulated inhibitor kinase (HRI), RNA-dependent protein kinase (PKR), and PKR-like endoplasmic reticulum kinase (PERK). These enzymes contain a similar catalytic domain allowing them to phosphorylate the same substrate, but have different accessory domains that regulate kinase activation in response to different signals. In GCN2 the functional kinase domain is followed by a histidyl-tRNA synthetase (HisRS)-like domain , which is the major motif for sensing amino acid starvation and triggering kinase activation; PERK has a transmembrane domain allowing it to reside in the endoplasmic reticulum membrane; the N-terminal domain can protrude into the lumen of the ER to sense unfolded proteins, while the catalytic domain extends into the cytoplasm where its substrate and effector mechanism lie; human PKR contains an RNA binding domain and responds to viral infection; and HRI contains haem binding sites to modulate translation of globin chains according to the availability of haem. In this way the eIF2α kinases can integrate diverse stress signals into a common pathway [12–14, 19].
Translation initiation requires the assembly of the 80S ribosome on the mRNA, which is mediated by proteins known as eukaryotic initiation factors (eIFs). Formation of the 43S pre-initiation complex depends on binding of the ternary complex that consists of the heterotrimeric G-protein eIF2 (α, β and γ subunits), methionyl-initiator tRNA (met-tRNAi) and GTP . Initiation of translation and release of the initiation factors involves hydrolysis of GTP to GDP, which leaves an inactive eIF2-GDP complex. Before further rounds of translation initiation can occur eIF2 must be reactivated by exchange of GDP for GTP . The presence of a phosphate group on the α subunit of eIF2 inhibits recycling of inactive eIF2-GDP to active eIF2-GTP by limiting the activity of the guanine nucleotide exchange factor, eIF2B . The consequence of activity of the eIF2α kinases therefore is global translation repression, since initiation complexes cannot form. In spite of the generalized reduction in translation, selected mRNAs are translated, whose products shapes the subsequent stress response. Reduced translation conserves energy and nutrients, allowing time for the cell to adapt appropriately to the stress conditions. This mechanism is conserved in the vast majority of eukaryotes. One notable exception is the Microsporidium Encephalitozoon cuniculi, whose kinome does not include eIF2α kinases (or other stress-response kinases), a probable adaptation to its parasitic lifestyle . It is, therefore, of interest to investigate the extent to which malaria parasites may rely on eIF2α phosphorylation for stress-response and/or life cycle progression.
A cluster of three sequences that includes PfPK4, a protein kinase that was previously described as a putative eIF2α kinase , was identified in the P. falciparum kinome on the basis of catalytic domain similarity [10, 23]. Here, evidence is provided that the P. falciparum eIF2α orthologue is phosphorylated in response to amino acid starvation. Bioinformatics analysis reveals that P. falciparum encodes three eIF2α kinases, one of which, P
e ukaryotic I nitiation Factor K inase- 1 (PfeIK1), is indeed be able to phosphorylate P. falciparum eIF2α in vitro. Reverse genetics experiments show that inactivation of the pfeik1 gene does not affect asexual growth, gametocytogenesis or further sexual development, since pfeik1
- sporozoites can be formed in the mosquito vector; in contrast, pfeik1
- parasites are unable to phosphorylate eIF2α in response to amino-acid starvation.