ABSTRACT
The association of the pellicle with cytoskeletal elements in Toxoplasma gondii allows this parasite to maintain its mechanical integrity and makes possible its gliding motility and cell invasion. The inner membrane complex (IMC) resembles the flattened membrane sacs observed in free-living protozoa and these sacs have been found to associate with cytoskeletal proteins such as articulins. We used immunofluorescence microscopy to characterise the presence and distribution of plateins, a sub-family of articulins, in T. gondii tachyzoites. A dispersed labelling of the whole protozoan body was observed. Electron microscopy of detergent-extracted cells revealed the presence of a network of 10 nm filaments distributed throughout the parasite. These filaments were labelled with anti-platein antibodies. Screening the sequenced T. gondii genome, we obtained the sequence of an IMC predicted protein with 25 percent identity and 42 percent similarity to the platein isoform alpha 1 present in Euplotes aediculatus, but with 42 percent identity and 55 percent similarity to that found in Euglena gracilis, suggesting strong resemblance to articulins.
Subject(s)
Cytoskeletal Proteins , Cytoskeleton , Protozoan Proteins , Toxoplasma , Base Sequence , Cytoskeletal Proteins/chemistry , Cytoskeletal Proteins/genetics , Cytoskeleton/chemistry , Cytoskeleton/ultrastructure , Microscopy, Electron , Microscopy, Immunoelectron , Molecular Sequence Data , Protozoan Proteins/chemistry , Protozoan Proteins/genetics , Sequence Alignment , Toxoplasma/genetics , Toxoplasma/metabolism , Toxoplasma/ultrastructureABSTRACT
The dense granule of Toxoplasma gondii is a secretory vesicular organelle of which the proteins participate in the modification of the parasitophorous vacuole (PV) and PV membrane for the maintenance of intracellular parasitism in almost all nucleated host cells. In this review, the archives on the research of GRA proteins are reviewed on the foci of finding GRA proteins, characterizing molecular aspects, usefulness in diagnostic antigen, and vaccine trials in addition to some functions in host-parasite interactions.
Subject(s)
Animals , Humans , Antigens, Protozoan/metabolism , Cytoplasmic Granules/metabolism , Host-Parasite Interactions , Protozoan Proteins/metabolism , Toxoplasma/metabolism , Toxoplasmosis/parasitology , Vacuoles/metabolismABSTRACT
Toxoplasma gondii is an intracellular obligate protozoan, which infects humans and warm-blooded animals. The aim of the present study was to clone the rop2, gra5 and gra7 genes from T. gondii RH strain and to produce recombinant proteins. The rop2, gra5 and gra7 gene fragments produced by polymerase chain reaction were cloned into the pET102/D-TOPO® vector which contains thioredoxin and polyhistidine tags at the C- and N-ends, respectively, and is expressed in Escherichia coli BL21(DE-3). The expression fusion proteins were found almost entirely in the insoluble form in the cell lysate. These recombinant proteins were purified with an Ni-NTA column. Concentrations of the recombinant antigens produced in the E. coli BL21-star ranged from 300 to 500 μg/ml growth media, which was used to immunize rabbits. We observed an identity ranging from 96 to 97% when nucleotide sequences were compared to GenBank database sequences. Immunocharacterization of proteins was made by indirect immunofluorescence assay. These proteins will be used for serodiagnosis and vaccination.
Subject(s)
Animals , Antigens, Protozoan/genetics , Membrane Proteins/genetics , Protozoan Proteins/genetics , Toxoplasma/genetics , Antigens, Protozoan/immunology , Antigens, Protozoan/metabolism , Blotting, Western , Cloning, Molecular , Electrophoresis, Polyacrylamide Gel , Fluorescent Antibody Technique, Indirect , Oligonucleotide Array Sequence Analysis , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Membrane Proteins/immunology , Membrane Proteins/metabolism , Protozoan Proteins/immunology , Protozoan Proteins/metabolism , Toxoplasma/immunology , Toxoplasma/metabolismABSTRACT
Interactions between GRA proteins of dense granules in Toxoplasma gondii and host cell proteins were analyzed by yeast two-hybrid technique. The cMyc-GRA fusion proteins expressed from pGBKT7 plasmid in Y187 yeast were bound to host cell proteins from pGADT7-Rec-HeLa cDNA library transformed to AH109 yeast by mating method. By the selection procedures, a total of 939 colonies of the SD/-AHLT culture, 348 colonies of the X-alpha-gal positive and PCR, 157 colonies of the X-beta-gal assay were chosen for sequencing the cDNA and finally 90 colonies containing ORF were selected to analyze the interactions. GRA proteins interacted with a variety of host cell proteins such as enzymes, structural and functional proteins of organellar proteins of broad spectrum. Several specific bindings of each GRA protein to host proteins were discussed presumptively the role of GRA proteins after secreting into the parasitophorous vacuoles (PV) and the PV membrane in the parasitism of this parasite.
Subject(s)
Humans , Animals , Vacuoles/metabolism , Two-Hybrid System Techniques , Toxoplasma/metabolism , Protozoan Proteins/metabolism , Proteins/metabolism , Organelles/metabolism , Intracellular Membranes/metabolism , HeLa Cells , Gene Library , Cytoplasmic GranulesABSTRACT
We are investigating the structure and biosynthesis of glycosyl-phosphatidylinositols (GPI) in the protozoa Toxoplasma gondii, Plasmodium falciparum, Plasmodium yoelii and Paramecium primaurelia. This comparison of structural and biosynthesis data should lead us to common and individual features of the GPI-biosynthesis and transport in different organisms.