title: TgGRA23, a novel Toxoplasma gondii dense granule protein associated with the parasitophorous vacuole membrane and intravacuolar network.
abstract: Toxoplasma gondii is an intracellular protozoan parasite, which relies on a specialized compartment, the parasitophorous vacuole (PV), to survive within host cells. Dense granules within the parasite release a large variety of proteins to maintain the integrity of the vacuole structure. Here, we identified a novel dense granule protein in T. gondii, TgGRA23, which is a homolog of the Sarcocystis muris dense granule protein, SmDG32. Recombinant TgGRA23 (rTgGRA23) expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein was used to raise antisera in mice and rabbits. Immunoblotting showed that antisera from the immunized mice and rabbits reacted with parasite lysates to yield a 21-kDa native protein. In addition, immuno-electron microscopic examination showed that TgGRA23 resides in the dense granules, PV membrane and intravacuolar network of the parasite. To confirm the precise subcellular localization of TgGRA23 in T. gondii, an immunofluorescent antibody test was performed using dense granule markers. Notably, TgGRA23 co-localized with other dense granule proteins including TgGRA4 and TgGRA7, in the extracellular-stage parasites. Biochemical experiments indicated that TgGRA23 is insoluble and may form an electrostatic complex that is resistant to non-ionic detergents. Furthermore, specific antibodies to TgGRA23 were detected during the chronic stage of Toxoplasma infection in mice. Our results suggest that TgGRA23 is an as yet unknown member of the T. gondii dense granule proteins, and that it may be involved in remodeling or maintenance of the PV.
authors.affiliation: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan.
authors.forename: Tatsunori
authors.initials: T
authors.lastname: Masatani
authors.forename: Tomohide
authors.initials: T
authors.lastname: Matsuo
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Tanaka
authors.forename: Mohamad Alaa
authors.initials: MA
authors.lastname: Terkawi
authors.forename: Eung-Goo
authors.initials: EG
authors.lastname: Lee
authors.forename: Youn-Kyoung
authors.initials: YK
authors.lastname: Goo
authors.forename: Gabriel Oluga
authors.initials: GO
authors.lastname: Aboge
authors.forename: Junya
authors.initials: J
authors.lastname: Yamagishi
authors.forename: Kei
authors.initials: K
authors.lastname: Hayashi
authors.forename: Kyohko
authors.initials: K
authors.lastname: Kameyama
authors.forename: Shinuo
authors.initials: S
authors.lastname: Cao
authors.forename: Yoshifumi
authors.initials: Y
authors.lastname: Nishikawa
authors.forename: Xuenan
authors.initials: X
authors.lastname: Xuan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000953:Antigens, Protozoan / Q000378:metabolism; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D005982:Glutathione Transferase / Q000235:genetics; D007106:Immune Sera / Q000276:immunology; D051379:Mice; D008813:Mice, Inbred ICR; D015800:Protozoan Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism; D016415:Sequence Alignment; D014122:Toxoplasma / Q000276:immunology / Q000378:metabolism*; D014123:Toxoplasmosis / Q000276:immunology*; D014617:Vacuoles / Q000737:chemistry / Q000378:metabolism; D014709:Vero Cells
chemical_list: D000953:Antigens, Protozoan; D007106:Immune Sera; D015800:Protozoan Proteins; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase