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Published on: February 8, 2012
[Secretion and Distribution of Rhoptry Protein 16 during Toxoplasma gondii Invasion into Host Cells]
Objective:
To examine the secretion and localization of Toxoplasma gondii Rhoptry protein 16 （ROP16） during invasion of different strains of T. gondii into host cells.
Methods:
The Tgrop16 gene was amplified by PCR on the cDNA of T. gondii RH strain, subcloned into the plasmid pET-32a（+）, and expressed in Escherichia coli BL21（DE3） under the induction of isopropyl β-D-1-thiogalactopyranoside. New Zealand rabbit was immuned with the expressed recombinant protein TgROP16 to produce polyclonal anti-TgROP16 antibody. The specificity and sensitivity of the polyclonal antibody were examined by Western blotting and indirect ELISA, respectively. The transcriptional and protein levels of Tgrop16 in T. gondii RH strain and Pru strain were determined by real-time PCR and Western blotting, respectively. The secretion and distribution of TgROP16 in human foreskin fibroblasts (HFFs) during the invasion by T. gondii RH strain and Pru strain were examined by indirect immunofluorescence assay (IFA).
Results:
Western blotting showed a specific band at M(r) of ～100 000, indicating that the specific rabbit-derived anti-TgROP16 polyclonal antibody was capable of recognizing TgROP16. Indirect ELISA revealed a titer of 1:25 600 for the antibody. The relative expression level of Tgrop16 in Pru strain［（7.786±0.206）］ was 7 times than that in RH strain［（1.000±0.110）］（P<0.05） as detected by real-time PCR, and TgROP16 protein level was higher in RH strain than in Pru strain. IFA showed that TgROP16 was localized on the apical complex of the unrecruited tachyzoite of T. gondii before invasion and was secreted out of the recruited tachyzoite after invasion.
Conclusion:
The anti-TgROP16 polyclonal antibody has high specificity and sensitivity. The TgROP16 protein level is higher in the RH strain than in the Pru strain. For both strains, TgROP16 is localized on the apical complex of the unrecruited tachyzoite before invasion and secreted out of the recruited tachyzoite during invasion.
Insights
Toxoplasma gondii Rhoptry protein 16 (ROP16) is secreted during host cell invasion. This study developed a specific antibody to track ROP16, finding it localized apically before invasion and secreted afterward in both RH and Pru strains.
Area of Science:
- Parasitology
- Molecular Biology
- Cell Biology
Background:
- Toxoplasma gondii is an obligate intracellular parasite.
- Rhoptry proteins are crucial for host cell invasion.
- ROP16 plays a role in T. gondii pathogenesis.
Purpose of the Study:
- To investigate the secretion and localization of Toxoplasma gondii Rhoptry protein 16 (ROP16).
- To compare ROP16 behavior during invasion by different T. gondii strains (RH and Pru) into host cells.
Main Methods:
- Amplification and expression of the Tgrop16 gene in E. coli.
- Production and validation of a rabbit polyclonal anti-TgROP16 antibody using Western blotting and ELISA.
- Quantification of Tgrop16 transcript and protein levels via real-time PCR and Western blotting.
- Examination of ROP16 secretion and localization using indirect immunofluorescence assay (IFA) in human foreskin fibroblasts (HFFs).
Main Results:
- A specific polyclonal antibody against TgROP16 was successfully generated with high specificity and sensitivity (titer 1:25,600).
- Real-time PCR indicated Tgrop16 transcript levels were approximately 7-fold higher in the Pru strain compared to the RH strain (P<0.05).
- Western blotting revealed higher TgROP16 protein levels in the RH strain than in the Pru strain.
- IFA demonstrated that TgROP16 is located on the apical complex of uninvaded tachyzoites and is secreted from the tachyzoite upon host cell invasion for both strains.
Conclusions:
- The developed anti-TgROP16 antibody is a reliable tool for studying ROP16.
- Differential expression of TgROP16 occurs between T. gondii RH and Pru strains.
- ROP16 exhibits conserved localization and secretion patterns during host cell invasion by different T. gondii strains.
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