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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abdoli A, Dalimi A, Movahedin M (2012) Impaired reproductive function of male rats infected with Toxoplasma gondii. Andrologia 44(Suppl 1):679–87. doi:10.1111/j.1439-0272.2011.01249.x
Achbarou A, Mercereau-Puijalon O, Autheman JM, Fortier B, Camus D, Dubremetz JF (1991) Characterization of microneme proteins of Toxoplasma gondii. Mol Biochem Parasitol 47(2):223–33
Ahn VE, Chu ML, Choi HJ, Tran D, Abo A, Weis WI (2011) Structural basis of Wnt signaling inhibition by Dickkopf binding to LRP5/6. Dev Cell 21(5):862–73. doi:10.1016/j.devcel.2011.09.003
Anantharaman V, Iyer LM, Balaji S, Aravind L (2007) Adhesion molecules and other secreted host-interaction determinants in Apicomplexa: insights from comparative genomics. Int Rev Cytol 262:1–74. doi:10.1016/S0074-7696(07)62001-4
Ang SJ, Stump RJ, Lovicu FJ, McAvoy JW (2004) Spatial and temporal expression of Wnt and Dickkopf genes during murine lens development. Gene Expr Patterns 4(3):289–95. doi:10.1016/j.modgep.2003.11.002
Arantes TP, Lopes WD, Ferreira RM, Pieroni JS, Pinto VM, Sakamoto CA, Costa AJ (2009) Toxoplasma gondii: evidence for the transmission by semen in dogs. Exp Parasitol 123(2):190–4. doi:10.1016/j.exppara.2009.07.003
Barragan A, Brossier F, Sibley LD (2005) Transepithelial migration of Toxoplasma gondii involves an interaction of intercellular adhesion molecule 1 (ICAM-1) with the parasite adhesin MIC2. Cell Microbiol 7(4):561–8. doi:10.1111/j.1462-5822.2005.00486.x
Carruthers VB, Boothroyd JC (2007) Pulling together: an integrated model of Toxoplasma cell invasion. Curr Opin Microbiol 10(1):83–9. doi:10.1016/j.mib.2006.06.017
Carruthers VB, Tomley FM (2008) Microneme proteins in apicomplexans. Subcell Biochem 47:33–45
Cerede O, Dubremetz JF, Bout D, Lebrun M (2002) The Toxoplasma gondii protein MIC3 requires pro-peptide cleavage and dimerization to function as adhesin. EMBO J 21(11):2526–36. doi:10.1093/emboj/21.11.2526
Cerede O, Dubremetz JF, Soete M, Deslee D, Vial H, Bout D, Lebrun M (2005) Synergistic role of micronemal proteins in Toxoplasma gondii virulence. J Exp Med 201(3):453–63. doi:10.1084/jem.20041672
Chu J, Zhang Z, Zheng Y, Yang J, Qin L, Lu J, Huang ZL, Zeng S, Luo Q (2009) A novel far-red bimolecular fluorescence complementation system that allows for efficient visualization of protein interactions under physiological conditions. Biosens Bioelectron 25(1):234–9. doi:10.1016/j.bios.2009.06.008
Fu JJ, Lu GX, Li LY, Liu G, Xing XW, Liu SF (2003) Molecular cloning for testis spermatogenesis cell apoptosis related gene TSARG1 and Mtsarg1 and expression analysis for Mtsarg1 gene. Yi Chuan Xue Bao 30(1):25–9
Gage PJ, Qian M, Wu D, Rosenberg KI (2008) The canonical Wnt signaling antagonist DKK2 is an essential effector of PITX2 function during normal eye development. Dev Biol 317(1):310–24. doi:10.1016/j.ydbio.2008.02.030
Garcia-Reguet N, Lebrun M, Fourmaux MN, Mercereau-Puijalon O, Mann T, Beckers CJ, Samyn B, Van Beeumen J, Bout D, Dubremetz JF (2000) The microneme protein MIC3 of Toxoplasma gondii is a secretory adhesin that binds to both the surface of the host cells and the surface of the parasite. Cell Microbiol 2(4):353–64
Harper JM, Hoff EF, Carruthers VB (2004) Multimerization of the Toxoplasma gondii MIC2 integrin-like A-domain is required for binding to heparin and human cells. Mol Biochem Parasitol 134(2):201–12. doi:10.1016/j.molbiopara.2003.12.001
Jiang H, Li LY, Lu GX (2001) Molecular cloning of genes related to apoptosis in spermatogenic cells of mouse. Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao Acta Biochimica et Biophysica Sinica 33(4):421–425
Jiang H, Li L, Lu GX (2009) Molecular cloning and characterization of a novel transcript variant of Mtsarg1 gene. Mol Biol Rep 36(5):1023–32. doi:10.1007/s11033-008-9276-6
Liu SG, Qin C, Yao ZJ, Wang D (2006) Study on the transmission of Toxoplasma gondii by semen in rabbits. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi 24(3):166–70
MacDonald BT, Tamai K, He X (2009) Wnt/beta-catenin signaling: components, mechanisms, and diseases. Dev Cell 17(1):9–26. doi:10.1016/j.devcel.2009.06.016
Mao B, Wu W, Li Y, Hoppe D, Stannek P, Glinka A, Niehrs C (2001) LDL-receptor-related protein 6 is a receptor for Dickkopf proteins. Nature 411(6835):321–5. doi:10.1038/35077108
Meissner M, Reiss M, Viebig N, Carruthers VB, Toursel C, Tomavo S, Ajioka JW, Soldati D (2002) A family of transmembrane microneme proteins of Toxoplasma gondii contain EGF-like domains and function as escorters. J Cell Sci 115(Pt 3):563–74
Monaghan AP, Kioschis P, Wu W, Zuniga A, Bock D, Poustka A, Delius H, Niehrs C (1999) Dickkopf genes are co-ordinately expressed in mesodermal lineages. Mech Dev 87(1–2):45–56
Montoya JG, Liesenfeld O (2004) Toxoplasmosis. Lancet 363(9425):1965–76. doi:10.1016/S0140-6736(04)16412-X
Mukhopadhyay M, Gorivodsky M, Shtrom S, Grinberg A, Niehrs C, Morasso MI, Westphal H (2006) Dkk2 plays an essential role in the corneal fate of the ocular surface epithelium. Development 133(11):2149–54. doi:10.1242/dev.02381
Muniz-Feliciano L, Van Grol J, Portillo JA, Liew L, Liu B, Carlin CR, Carruthers VB, Matthews S, Subauste CS (2013) Toxoplasma gondii-induced activation of EGFR prevents autophagy protein-mediated killing of the parasite. PLoS Pathog 9(12):e1003809. doi:10.1371/journal.ppat.1003809
Nie X (2005) Dkk1, −2, and −3 expression in mouse craniofacial development. J Mol Histol 36(5):367–72. doi:10.1007/s10735-005-9008-3
Niehrs C (2006) Function and biological roles of the Dickkopf family of Wnt modulators. Oncogene 25(57):7469–81. doi:10.1038/sj.onc.1210054
Nusse R (2005) Wnt signaling in disease and in development. Cell Res 15(1):28–32. doi:10.1038/sj.cr.7290260
Printen JA, Sprague GF Jr (1994) Protein-protein interactions in the yeast pheromone response pathway: Ste5p interacts with all members of the MAP kinase cascade. Genetics 138(3):609–19
Santana LF, da Costa AJ, Pieroni J, Lopes WD, Santos RS, de Oliveira GP, de Mendonca RP, Sakamoto CA (2010) Detection of Toxoplasma gondii in the reproductive system of male goats. Rev Bras Parasitol Vet 19(3):179–82
Sharma P, Chitnis CE (2013) Key molecular events during host cell invasion by apicomplexan pathogens. Curr Opin Microbiol 16(4):432–7. doi:10.1016/j.mib.2013.07.004
Sun LH, Fan F, Wang JJ, Gong J (2008) Acute Toxoplasma gondii infection affects the reproductive function of male mice. Zhonghua Nan Ke Xue 14(1):55–7
Tao Q, Fang R, Zhang W, Wang Y, Chen J, Li Y, Fang K, Khan MK, Hu M, Zhou Y, Zhao J (2013) Protective immunity induced by a DNA vaccine-encoding Toxoplasma gondii microneme protein 11 against acute toxoplasmosis in BALB/c mice. Parasitol Res 112(8):2871–7. doi:10.1007/s00436-013-3458-4
Tenter AM, Heckeroth AR, Weiss LM (2000) Toxoplasma gondii: from animals to humans. Int J Parasitol 30(12–13):1217–58
Terpsidis KI, Papazahariadou MG, Taitzoglou IA, Papaioannou NG, Georgiadis MP, Theodoridis IT (2009) Toxoplasma gondii: reproductive parameters in experimentally infected male rats. Exp Parasitol 121(3):238–41. doi:10.1016/j.exppara.2008.11.006
Tomley FM, Soldati DS (2001) Mix and match modules: structure and function of microneme proteins in apicomplexan parasites. Trends Parasitol 17(2):81–8
Wang Y, Fang R, Yuan Y, Hu M, Zhou Y, Zhao J (2014) Identification of host proteins interacting with the integrin-like A domain of Toxoplasma gondii micronemal protein MIC2 by yeast-two-hybrid screening. Parasit Vectors 7(1):543. doi:10.1186/PREACCEPT-1400249986144212
Wright HT, Sandrasegaram G, Wright CS (1991) Evolution of a family of N-acetylglucosamine binding proteins containing the disulfide-rich domain of wheat germ agglutinin. J Mol Evol 33(3):283–94
Zorn AM (2001) Wnt signalling: antagonistic Dickkopfs. Curr Biol 11(15):R592–5
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).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
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.
Authors’ contributions
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.
Rights and permissions
About this article
Cite this article
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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00436-016-5033-2