The ubiquitous intracellular parasite Toxoplasma gondii (phylum Apicomplexa) differentiates into an encysted form (bradyzoite) that can repeatedly re-emerge as a life-threatening acute infection (tachyzoite) upon impairment of immunity. Since the switch from tachyzoite to bradyzoite is a stress-induced response, we sought to identify components related to the phosphorylation of the α subunit of eIF2 (eukaryotic initiation factor-2), a well-characterized event associated with stress remediation in other eukaryotic systems. In addition to characterizing Toxoplasma eIF2α (TgIF2α), we have discovered a novel eIF2 protein kinase, designated TgIF2K-A (Toxoplasma gondiiinitiation factor-2kinase). Although the catalytic domain of TgIF2K-A contains sequence and structural features that are conserved among members of the eIF2 kinase family, TgIF2K-A has an extended N-terminal region that is highly divergent from other eIF2 kinases. TgIF2K-A specifically phosphorylates the regulatory serine residue of yeast eIF2α in vitro and in vivo, and can modulate translation when expressed in the yeast model system. We also demonstrate that TgIF2K-A phosphorylates the analogous regulatory serine residue of recombinant TgIF2α in vitro. Finally, we demonstrate that TgIF2α phosphorylation in tachyzoites is enhanced in response to heat shock or alkaline stress, conditions known to induce parasite differentiation in vitro. Collectively, this study suggests that eIF2 kinase-mediated stress responses are conserved in Apicomplexa, and a novel family member exists that may control parasite-specific events, including the clinically relevant conversion into bradyzoite cysts.
Abbreviations used: ATF(4), activating transcription factor (4); bZIP, basic zipper; eIF2, eukaryotic initiation factor-2; GCN(2), general control of non-derepressible (2); GST, glutathione S-transferase; HRI, haem-regulated inhibitor; ORF, open reading frame; PEK, pancreatic eIF-2α kinase; PKR, double-stranded-RNA-dependent protein kinase; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase; TgIF2α, Toxoplasma gendii eIF2α; UTR, untranslated region.
The nucleotide sequence data reported will appear in DDBJ, EMBL, GenBank® and GSDB Nucleotide Sequence Databases under the accession numbers AY518936 and AY518935 for TgIF2K-A and TgIF2α respectively.