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.
Vacuoles/*metabolism ; Two-Hybrid System Techniques ; Toxoplasma/metabolism/*pathogenicity ; Protozoan Proteins/*metabolism/secretion ; Proteins/*metabolism ; Organelles/metabolism ; Intracellular Membranes/*metabolism ; Humans ; Hela Cells ; Gene Library ; Cytoplasmic Granules ; Animals

BACKGROUNDAlthough the onset of anemia during infectious disease is commonly correlated with production of inflammatory cytokines, the mechanisms by which cytokines induce anemia are poorly defined. This study focused on the mechanism research.

METHODSDifferent types of mice were infected perorally with Toxoplasma gondii strain ME49. At the indicated times, samples from each mouse were harvested, processed, and analyzed individually. Blood samples were analyzed using a Coulter Counter and red blood cell (RBC) survival was measured by biotinylation. Levels of tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS), and inducible protein 10 (IP-10) mRNA in liver tissue were measured by real-time polymerase chain reaction.

RESULTST. gondii-infected mice exhibited anemia due to a decrease in both erythropoiesis and survival time of RBC in the circulation (P < 0.02). In addition, infection-stimulated anemia was associated with fecal occult, supporting previous literature that hemorrhage is a consequence of T. gondii infection in mice. Infection-induced anemia was abolished in interferon gamma (IFNγ) and IFNγ receptor deficient mice (P < 0.05) but was still evident in mice lacking TNF-α, iNOS, phagocyte NADPH oxidase or IP-10 (P < 0.02). Neither signal transducer and activator of transcription 1 (STAT1) deficient mice nor 129S6 controls exhibited decreased erythropoiesis, but rather suffered from an anemia resulting solely from increased loss of circulating RBC.

CONCLUSIONSInfection-stimulated decrease in erythropoiesis and losses of RBC have distinct mechanistic bases. These results show that during T. gondii infection, IFNγ is responsible for an anemia that results from both a decrease in erythropoiesis and a STAT1 independent loss of circulating RBC.

Anemia ; genetics ; metabolism ; Animals ; Erythrocytes ; pathology ; Interferon-gamma ; metabolism ; Male ; Mice ; Mice, Knockout ; Nitric Oxide Synthase Type II ; genetics ; metabolism ; Receptors, Interferon ; genetics ; metabolism ; STAT1 Transcription Factor ; genetics ; metabolism ; Toxoplasma ; pathogenicity ; Tumor Necrosis Factor-alpha ; genetics ; metabolism