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Updated: Apr 30, 2026
A Genetic Screen to Isolate Toxoplasma gondii Host-cell Egress Mutants
Published on: February 8, 2012
[Screening of host cell proteins that interact with Toxoplasma gondii ROP18 via yeast two-hybrid system]
Objective:
To screen the host cell proteins that can interact with Toxoplasma gondii ROP18 by using yeast two-hybrid system.
Methods:
The ROP18 gene fragments were amplified by RT-PCR from mRNA of T. gondii RH strain. The product of RT-PCR was digested with double restriction enzyme and was subcloned into the bait vector pGBKT7. The recombinant plasmid was transferred into yeast AH109 strain. Its toxicity and the autonomous activating activity were tested. The human fetal brain cDNA library was screened with pGBKT7-ROP18(25-251aa) as the bait plasmid by yeast two-hybrid system.
Results:
The bait was constructed and AH109/PGBKT7-ROP18 showed an autonomous activity. The yeast strain AH109/pGBKT7-ROP18(25-251aa) line was then mated with the Mate & PlateTM Human Fetal Brain cDNA library. Using the selection procedures, eight novel host cell proteins were obtained: damage-specific DNA binding protein 1 (DDB1), torsin A interacting protein 1 (TOR1AIP1), integrin beta 1, solute carrier family 3 (SLC3A2), tyrosyl protein sulfotransferase (TPST2), OCIA domain containing 1 (OCIAD1), Derl-like domain family member 2 (DERL2), in addition to Homo sapiens activating transcription factor 6 beta(ATF6).
Conclusion:
Eight novel host cell proteins have been obtained via yeast two-hybrid system, which can interact with TgROP18.
Insights
This study identified eight host cell proteins interacting with Toxoplasma gondii ROP18 using a yeast two-hybrid system. These findings advance understanding of host-pathogen interactions in toxoplasmosis.
Area of Science:
- Molecular Biology
- Parasitology
- Host-Pathogen Interactions
Background:
- Toxoplasma gondii ROP18 is a key virulence factor.
- Understanding host cell protein interactions is crucial for T. gondii pathogenesis.
Purpose of the Study:
- To identify host cell proteins that interact with T. gondii ROP18.
- To utilize the yeast two-hybrid system for screening protein interactions.
Main Methods:
- Amplification of ROP18 gene fragments via RT-PCR.
- Cloning into a yeast two-hybrid bait vector (pGBKT7).
- Screening a human fetal brain cDNA library using the yeast two-hybrid system.
Main Results:
- Successfully constructed the ROP18 bait plasmid.
- Identified eight novel host cell proteins interacting with T. gondii ROP18.
- These proteins include DDB1, TOR1AIP1, integrin beta 1, SLC3A2, TPST2, OCIAD1, DERL2, and ATF6.
Conclusions:
- The yeast two-hybrid system effectively identified host proteins interacting with TgROP18.
- Eight novel interacting host proteins were discovered, providing new targets for research.
