Interaction between Parasitophorous Vacuolar Membrane-associated GRA3 and Calcium Modulating Ligand of Host Cell Endoplasmic Reticulum in the Parasitism of Toxoplasma gondii

Abstract

A monoclonal antibody against Toxoplasma gondii of Tg556 clone (Tg556) blotted a 29 kDa protein, which was localized in the dense granules of tachyzoites and secreted into the parasitophorous vacuolar membrane (PVM) after infection to host cells. A cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg556, and the full-length was completed by 5′ -RACE of 2,086 bp containing an open reading frame (ORF) of 669 bp. The ORF encoded a polypeptide of 222 amino acids homologous to the revised GRA3 but not to the first reported one. The polypeptide has 3 hydrophobic moieties of an N-terminal stop transfer sequence and 2 transmembrane domains (TMD) in posterior half of the sequence, a cytoplasmic localization motif after the second TMD and an endoplasmic reticulum (ER) retrival motif in the C-terminal end, which suggests GRA3 as a type III transmembrane protein. With the ORF of GRA3, yeast two-hybrid assay was performed in HeLa cDNA expression library, which resulted in the interaction of GRA3 with calcium modulating ligand (CAMLG), a type II transmembrane protein of ER. The specific binding of GRA3 and CAMLG was confirmed by glutathione S-transferase (GST) pull-down and immunoprecipitation assays. The localities of fluorescence transfectionally expressed from GRA3 and CAMLG plasmids were overlapped completely in HeLa cell cytoplasm. In immunofluorescence assay, GRA3 and CAMLG were shown to be co-localized in the PVM of host cells. Structural binding of PVM-inserted GRA3 to CAMLG of ER suggested the receptor-ligand of ER recruitment to PVM during the parasitism of T. gondii.

Host Organelle Hijackers: a similarmodus operandiforToxoplasma gondiiandChlamydia trachomatis: co-infection model as a tool to investigate pathogenesis

“…20 nm), and ribosomes are restricted to the opposite face of the ER away from the PV. The retention of the ER at the PV has been proposed to be mediated by two parasite proteins that are anchored to the PV membrane: ROP2, which contains ER‐targeting domains exposed to the host cytosol and GRA3, which interacts with the host ER type II transmembrane protein calcium‐modulating ligand (CAMLG) (Sinai & Joiner, ; Kim et al ., ). By comparison, the interaction of the inclusion of C. trachomatis with the host ER is more intimate than for the Toxoplasma PV as the inclusion membrane forms direct membrane contact sites (MCSs or zones of close apposition < 50 nm) with ER elements (Derré et al ., ; Dumoux et al ., ).…”

Interactions of pathogen-containing compartments with the secretory pathway

“…Other T. gondii proteins from the dense granule (the GRA proteins including GRA3, GRA7, GRA8 and GRA14) have been shown to localize to the PVM where they can potentially interface with the ER and other host cell organelles (Mercier et al ., ; Rome et al ., ). Moreover, GRA3 was shown to directly interact and colocalize with the host ER protein calcium modulating ligand (CAMLG) providing a direct means of association between the two compartments (Kim et al ., ).…”

Neospora caninum Recruits Host Cell Structures to Its Parasitophorous Vacuole and Salvages Lipids from Organelles

“…In T. gondii (RH), host ER elements are closely apposed to the PV membrane (55). The molecular machinery of this process is still unknown, but two candidates have been proposed: ROP2, which contains an ER-targeting domain exposed to the host cytosol, and GRA3, which interacts with the host ER type II transmembrane protein calcium modulating ligand (CAMLG) (81,85). A GRA3 gene is present in the genome of N. caninum although transcriptional profiles show a strong downregulation of GRA3 in cultured Neospora.…”