Phosphoproteome of Toxoplasma gondii Infected Host Cells Reveals Specific Cellular Processes Predominating in Different Phases of Infection.

The invasion of Toxoplasma gondii tachyzoites into the host cell results in extensive host cell signaling activation/deactivation that is usually regulated by the phosphorylation/dephosphorylation. To elucidate how T. gondii regulates host cell signal transduction, the comparative phosphoproteome of stable isotope labeling with amino acids in cell culture-labeled human foreskin fibroblast cells was analyzed. The cells were grouped (Light [L], Medium [M], and Heavy [H] groups) based on the labeling isotope weight and were infected with T. gondii for different lengths of time (L: 0 hour; M: 2 hours; and H: 6 hours). A total of 892 phosphoproteins were identified with 1,872 phosphopeptides and 1,619 phosphorylation sites. The M versus L comparison revealed 694 significantly regulated phosphopeptides (436 upregulated and 258 downregulated). The H versus L comparison revealed 592 significantly regulated phosphopeptides (146 upregulated and 446 downregulated). The H versus M comparison revealed 794 significantly regulated phosphopeptides (149 upregulated and 645 downregulated). At 2 and 6 hours post-T. gondii infection, the most predominant host cell reactions were cell cycle regulation and cytoskeletal reorganization, which might be required for the efficient invasion and multiplication of T. gondii. Similar biological process profiles but different molecular function categories of host cells infected with T. gondii for 2 and 6 hours, which suggested that the host cell processes were not affected significantly by T. gondii infection but emphasized some differences in specific cellular processes at this two time points. Western blotting verification of some significantly regulated phosphoprotein phosphorylation sites was consistent with the mass spectra data. This study provided new insights into and further understanding of pathogen-host interactions from the host cell perspective.

[Observation of the Feline Intestinal Epithelial Cell Infected with Toxoplasma gondii].

Small intestine samples of neonatal cat were aseptically collected from the jejunum-ileum region and digested with collagenase XI/dispase I. Immunohistochemistry results showed that feline intestinal epithelial cells were successfully isolated and could be cultured. Cytokeratin was positive in the cytoplasm of feline intestinal epithelial cells. The cells were infected with the bradyzoites of Toxoplasma gondii Prugniaud strain, and the rupture of the cells was observed on the 72nd day post-infection. The sexual stage of T. gondii did not occur, however.

[Preparation and purification of polyclonal antibody against Toxoplasma gondii rhoptry protein 2 (ROP2) and its application in immunofluorescence localization].

OBJECTIVE: To prepare and purify polyclonal antibody against Toxoplasma gondii rhoptry protein 2 (ROP2) and apply it to immunofluorescence localization. METHODS: The constructed recombinant plasmid pET32a-ROP2 was transformed into E. coli BL21 (DE3) and the protein was expressed under the condition of 0.5 mmol/L IPTG induction. Cells were lysed by multiple rounds of sonication to obtain supernatant and inclusion body, respectively. The washed inclusion bodies were dissolved in urea. New Zealand White rabbits were immunized with 200 microg purified recombinant ROP2 mixing with the same volume of Freund's adjuvant for 3 times at interval of 14 days and 19 days, respectively. Rabbit serum was collected at 10 days after the last immunization. Polyclonal antibody in rabbit serum was purified with HiTrap Protein G HP affinity purification column. Indirect ELISA and Western blotting were used to detect antibody titer and specificity of polyclonal antibody against the recombinant ROP2. The polyclonal antibody was used to the localization of ROP2 on the parasitophorous vacuole membrane in human foreskin fibroblasts infected by Toxoplasma tachyzoites by the immunofluorescence method. RESULTS: The recombinant ROP2 protein was obtained and specific rabbit-derived polyclonal antibody was prepared. Indirect ELISA confirmed that the rabbit-derived polyclonal antibody titer reached 1:102400, and the recombinant ROP2 protein was recognized by specific polyclonal antibody. Immunofluorescence localization test showed that the ROP2 protein was located on the parasitophorous vacuole membrane. CONCLUSION: The rabbit-derived polyclonal antibody against ROP2 is prepared, and used in immunofluorescence localization of ROP2 on parasitophorous vacuole membrane.