Abstract
As an obligate intracellular protozoan, Toxoplasma gondii is a successful pathogen infecting a variety of animals, including humans. As an adhesin involving in host invasion, the micronemal protein MIC3 plays important roles in host cell attachment, as well as modulation of host EGFR signaling cascade. However, the specific host proteins that interact with MIC3 are unknown and the identification of such proteins will increase our understanding of how MIC3 exerts its functions. This study was designed to identify host proteins interacting with MIC3 by yeast two-hybrid screens. Using MIC3 as bait, a library expressing mouse proteins was screened, uncovering eight mouse proteins that showed positive interactions with MIC3. Two of which, spermatogenesis-associated protein 3 (Spata3) and dickkopf-related protein 2 (Dkk2), were further confirmed to interact with MIC3 by additional protein-protein interaction tests. The results also revealed that the tandem repeat EGF domains of MIC3 were critical in mediating the interactions with the identified host proteins. This is the first study to show that MIC3 interacts with host proteins that are involved in reproduction, growth, and development. The results will provide a clearer understanding of the functions of adhesion-associated micronemal proteins in T. gondii.
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Acknowledgement
The authors would like to acknowledge the generous help from members of the State Key Laboratory of Agricultural Microbiology. This work was supported by the National Natural Science Foundation of China (R.F., No. 31572510), National Key Basic Research Program (973 program) of China (J.Z., No. 2015CB150300), Huazhong Agricultural University Scientific and Technological Self-innovation Foundation (J.Z., No. 2662014BQ020) and Fundamental Research Funds for the Central Universities (No. 2662015PY048).
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All animal experiments were performed under the instructions and approval of Laboratory Animals Research Centre of Hubei province in People’s Republic of China and the ethics committee of Huazhong Agricultural University (Permit number: 4200695757). All female KunMing mice (4–6 weeks of age) were purchased from Laboratory Animals Research Centre of Hubei province. The mice were maintained under controlled conditions (temperature of 25 ± 1 °C) with instant access to feeding and water.
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JZ and RF conceived and designed the study and critically revised the manuscript. YW carried out all of the experiments and drafted the manuscript. YY and MP helped carry out yeast two-hybrid screening. MH, YZ, and BS contributed to the revision of the manuscript. All authors read and approved the final manuscript.
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Wang, Y., Fang, R., Yuan, Y. et al. Identification of host proteins, Spata3 and Dkk2, interacting with Toxoplasma gondii micronemal protein MIC3. Parasitol Res 115, 2825–2835 (2016). https://doi.org/10.1007/s00436-016-5033-2
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DOI: https://doi.org/10.1007/s00436-016-5033-2