title: Promastigote to amastigote differentiation of Leishmania is markedly delayed in the absence of PERK eIF2alpha kinase-dependent eIF2alpha phosphorylation.
abstract: The parasitic protozoan Leishmania is the etiological agent of human leishmaniasis worldwide. It undergoes cellular differentiation from the sandfly promastigote form into amastigotes within mammalian macrophages, a process that is essential for its intracellular survival. Here, we characterized the Leishmania infantum PERK eIF2alpha kinase homologue and addressed its role in the parasite's cytodifferentiation. We show that Leishmania PERK is an endoplasmic reticulum (ER) transmembrane protein that largely colocalizes with the ER BiP chaperone. The Leishmania PERK catalytic kinase domain undergoes autohyperphosphorylation and phosphorylates the translation initiation factor 2-alpha subunit (eIF2alpha) in vitro at threonine 166. We also report that PERK is post-translationally regulated specifically in the intracellular stage of the parasite or under ER stress, most likely through extensive autohyperphosphorylation. We have generated a PERK dominant negative mutant overexpressing a truncated PERK protein lacking the N-terminal luminal domain and showed that this mutant is impaired in eIF2alpha phosphorylation in response to ER stress or during amastigote differentiation. Most importantly, we showed that lack of eIF2alpha phosphorylation markedly delays the Leishmania differentiation process towards amastigote forms both in parasites grown axenically or within macrophages. These data highlight the importance of PERK eIF2alpha kinase-dependent eIF2alpha phosphorylation in the intracellular development of Leishmania.
authors.affiliation: Genera Biosystems, Small Technologies Cluster, 1 Dalmore Drive, Scoresby, Victoria 3179, Australia.
authors.forename: Conan
authors.initials: C
authors.lastname: Chow
authors.forename: Serge
authors.initials: S
authors.lastname: Cloutier
authors.forename: Carole
authors.initials: C
authors.lastname: Dumas
authors.forename: Marie-Noelle
authors.initials: MN
authors.lastname: Chou
authors.forename: Barbara
authors.initials: B
authors.lastname: Papadopoulou
mesh_terms: D000595:Amino Acid Sequence; D004721:Endoplasmic Reticulum / Q000201:enzymology; D015852:Eukaryotic Initiation Factor-2 / Q000378:metabolism*; D018314:Leishmania infantum / Q000201:enzymology* / Q000254:growth & development*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D019892:eIF-2 Kinase / Q000235:genetics / Q000378:metabolism*
chemical_list: D015852:Eukaryotic Initiation Factor-2; D050505:Mutant Proteins; D019892:eIF-2 Kinase