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Identification and characterization of a microneme protein (NcMIC6) in Neospora caninum

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Abstract

Neospora caninum, an Apicomplexa parasite, is the causative agent of neosporosis. As described for other members of Apicomplexa, microneme proteins (MICs) play a key role in attachment and invasion of host cells by N. caninum. Herein we identified N. caninum microneme protein 6 (NcMIC6) that is orthologous to Toxoplasma gondii microneme protein 6 (TgMIC6). The open reading frame of the NcMIC6 gene is 984 bp and encodes a 327 amino acid peptide. Sequence analysis showed that NcMIC6 included a signal peptide, a transmembrane region, three epidermal growth factor-like (EGF) domains, and two low complexity regions. Antibodies raised against recombinant NcMIC6 recognized an approximately 35-kDa native MIC6 protein in Western blots of N. caninum tachyzoites. Immunofluorescence analysis showed that NcMIC6 had a polar labeling pattern, which was consistent with localization of micronemes in the apical region. Pulse invasion assays showed that NcMIC6 translocated from the apical tip to the posterior end of the parasites. Secretion assays demonstrated that NcMIC6 was released into the supernatants. Importantly, it was clearly revealed by co-immunoprecipitation that NcMIC6 formed a complex with other two soluble microneme proteins (NcMIC1 and NcMIC4). In conclusion, identification and characterization of the novel microneme protein NcMIC6 may contribute to understanding how this protein functions during the parasite motility and host cell invasion.

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References

  • Alexander DL, Mital J, Ward GE, Bradley P, Boothroyd JC (2005) Identification of the moving junction complex of Toxoplasma gondii: a collaboration between distinct secretory organelles. PLoS Pathog 1, e17

    Article  PubMed Central  PubMed  Google Scholar 

  • Brecht S, Carruthers VB, Ferguson DJ, Giddings OK, Wang G, Jakle U, Harper JM, Sibley LD, Soldati D (2001) The toxoplasma micronemal protein MIC4 is an adhesin composed of six conserved apple domains. J Biol Chem 276:4119–4127

    Article  CAS  PubMed  Google Scholar 

  • Bumstead J, Tomley F (2000) Induction of secretion and surface capping of microneme proteins in Eimeria tenella. Mol Biochem Parasitol 110:311–321

    Article  CAS  PubMed  Google Scholar 

  • Carruthers V, Boothroyd JC (2007) Pulling together: an integrated model of Toxoplasma cell invasion. Curr Opin Microbiol 10:83–89

    Article  CAS  PubMed  Google Scholar 

  • Carruthers VB, Sibley LD (1997) Sequential protein secretion from three distinct organelles of Toxoplasma gondii accompanies invasion of human fibroblasts. Eur J Cell Biol 73:114–123

    CAS  PubMed  Google Scholar 

  • Carruthers VB, Sibley LD (1999) Mobilization of intracellular calcium stimulates microneme discharge in Toxoplasma gondii. Mol Microbiol 31:421–428

    Article  CAS  PubMed  Google Scholar 

  • Carruthers VB, Moreno SN, Sibley LD (1999) Ethanol and acetaldehyde elevate intracellular [Ca2+] and stimulate microneme discharge in Toxoplasma gondii. Biochem J 342(Pt 2):379–386

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Davis CG (1990) The many faces of epidermal growth factor repeats. New Biol 2:410–419

    CAS  PubMed  Google Scholar 

  • Dubey JP (2003) Review of Neospora caninum and neosporosis in animals. Korean J Parasitol 41:1–16

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Dubey JP, Lindsay DS (1996) A review of Neospora caninum and neosporosis. Vet Parasitol 67:1–59

    Article  CAS  PubMed  Google Scholar 

  • Dubey JP, Schares G, Ortega-Mora LM (2007) Epidemiology and control of neosporosis and Neospora caninum. Clin Microbiol Rev 20:323–367

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • El Hajj H, Papoin J, Cerede O, Garcia-Reguet N, Soete M, Dubremetz JF, Lebrun M (2008) Molecular signals in the trafficking of Toxoplasma gondii protein MIC3 to the micronemes. Eukaryot Cell 7:1019–1028

    Article  PubMed Central  PubMed  Google Scholar 

  • Harper JM, Huynh MH, Coppens I, Parussini F, Moreno S, Carruthers VB (2006) A cleavable propeptide influences Toxoplasma infection by facilitating the trafficking and secretion of the TgMIC2-M2AP invasion complex. Mol Biol Cell 17:4551–4563

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hehl AB, Lekutis C, Grigg ME, Bradley PJ, Dubremetz JF, Ortega-Barria E, Boothroyd JC (2000) Toxoplasma gondii homologue of plasmodium apical membrane antigen 1 is involved in invasion of host cells. Infect Immun 68:7078–7086

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hoff EF, Cook SH, Sherman GD, Harper JM, Ferguson DJ, Dubremetz JF, Carruthers VB (2001) Toxoplasma gondii: molecular cloning and characterization of a novel 18-kDa secretory antigen, TgMIC10. Exp Parasitol 97:77–88

    Article  CAS  PubMed  Google Scholar 

  • Huynh MH, Rabenau KE, Harper JM, Beatty WL, Sibley LD, Carruthers VB (2003) Rapid invasion of host cells by Toxoplasma requires secretion of the MIC2-M2AP adhesive protein complex. EMBO J 22:2082–2090

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Keller N, Naguleswaran A, Cannas A, Vonlaufen N, Bienz M, Bjorkman C, Bohne W, Hemphill A (2002) Identification of a Neospora caninum microneme protein (NcMIC1) which interacts with sulfated host cell surface glycosaminoglycans. Infect Immun 70:3187–3198

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Keller N, Riesen M, Naguleswaran A, Vonlaufen N, Stettler R, Leepin A, Wastling JM, Hemphill A (2004) Identification and characterization of a Neospora caninum microneme-associated protein NcMIC4 that exhibits unique lactose-binding properties. Infect Immun 72:4791–4800

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ling IT, Ogun SA, Momin P, Richards RL, Garcon N, Cohen J, Ballou WR, Holder AA (1997) Immunization against the murine malaria parasite Plasmodium yoelii using a recombinant protein with adjuvants developed for clinical use. Vaccine 15:1562–1567

    Article  CAS  PubMed  Google Scholar 

  • Lourenco EV, Pereira SR, Faca VM, Coelho-Castelo AA, Mineo JR, Roque-Barreira MC, Greene LJ, Panunto-Castelo A (2001) Toxoplasma gondii micronemal protein MIC1 is a lactose-binding lectin. Glycobiology 11:541–547

    Article  CAS  PubMed  Google Scholar 

  • Lovett JL, Howe DK, Sibley LD (2000) Molecular characterization of a thrombospondin-related anonymous protein homologue in Neospora caninum. Mol Biochem Parasitol 107:33–43

    Article  CAS  PubMed  Google Scholar 

  • 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 115:563–574

    CAS  PubMed  Google Scholar 

  • Moreno SN, Zhong L (1996) Acidocalcisomes in Toxoplasma gondii tachyzoites. Biochem J 313:655–659

    CAS  PubMed Central  PubMed  Google Scholar 

  • Naguleswaran A, Cannas A, Keller N, Vonlaufen N, Schares G, Conraths FJ, Bjorkman C, Hemphill A (2001) Neospora caninum microneme protein NcMIC3: secretion, subcellular localization, and functional involvement in host cell interaction. Infect Immun 69:6483–6494

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Naguleswaran A, Cannas A, Keller N, Vonlaufen N, Bjorkman C, Hemphill A (2002) Vero cell surface proteoglycan interaction with the microneme protein NcMIC3 mediates adhesion of Neospora caninum tachyzoites to host cells unlike that in Toxoplasma gondii. Int J Parasitol 32:695–704

    Article  CAS  PubMed  Google Scholar 

  • Rabenau KE, Sohrabi A, Tripathy A, Reitter C, Ajioka JW, Tomley FM, Carruthers VB (2001) TgM2AP participates in Toxoplasma gondii invasion of host cells and is tightly associated with the adhesive protein TgMIC2. Mol Microbiol 41:537–547

    Article  CAS  PubMed  Google Scholar 

  • Reiss M, Viebig N, Brecht S, Fourmaux MN, Soete M, Di Cristina M, Dubremetz JF, Soldati D (2001) Identification and characterization of an escorter for two secretory adhesins in Toxoplasma gondii. J Cell Biol 152:563–578

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Saouros S, Sawmynaden K, Marchant J, Simpson P, Matthews S (2008) Complete resonance assignment of the galectin-like domain of MIC1 from Toxoplasma gondii in complex with the second EGF domain from MIC6 and the backbone assignment in complex with the third EGF domain. Biomol NMR Assign 2:175–177

    Article  CAS  PubMed  Google Scholar 

  • Sawmynaden K, Saouros S, Marchant J, Simpson P, Matthews S (2008) Complete NMR assignments for the second EGF domain of MIC6 from Toxoplasma gondii and re-assignment in complex with the galectin-like domain of MIC1. Biomol NMR Assign 2:187–189

    Article  CAS  PubMed  Google Scholar 

  • Sheiner L, Santos JM, Klages N, Parussini F, Jemmely N, Friedrich N, Ward GE, Soldati D (2010) Toxoplasma gondii transmembrane microneme proteins and their modular design. Mol Microbiol 77:912–929

    CAS  PubMed Central  PubMed  Google Scholar 

  • Sonda S, Fuchs N, Gottstein B, Hemphill A (2000) Molecular characterization of a novel microneme antigen in Neospora caninum. Mol Biochem Parasitol 108:39–51

    Article  CAS  PubMed  Google Scholar 

  • Sultan AA, Thathy V, Frevert U, Robson KJ, Crisanti A, Nussenzweig V, Nussenzweig RS, Menard R (1997) TRAP is necessary for gliding motility and infectivity of plasmodium sporozoites. Cell 90:511–522

    Article  CAS  PubMed  Google Scholar 

  • Tranas J, Heinzen RA, Weiss LM, McAllister MM (1999) Serological evidence of human infection with the protozoan Neospora caninum. Clin Diagn Lab Immunol 6:765–767

    CAS  PubMed Central  PubMed  Google Scholar 

  • Tyler JS, Treeck M, Boothroyd JC (2011) Focus on the ringleader: the role of AMA1 in apicomplexan invasion and replication. Trends Parasitol 27:410–420

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Vieira MC, Moreno SN (2000) Mobilization of intracellular calcium upon attachment of Toxoplasma gondii tachyzoites to human fibroblasts is required for invasion. Mol Biochem Parasitol 106:157–162

    Article  CAS  PubMed  Google Scholar 

  • Yin J, Qu G, Cao L, Li Q, Fetterer R, Feng X, Liu Q, Wang G, Qi D, Zhang X, Miramontes E, Jenkins M, Zhang N, Tuo W (2012) Characterization of Neospora caninum microneme protein 10 (NcMIC10) and its potential use as a diagnostic marker for neosporosis. Vet Parasitol 187:28–35

    Article  CAS  PubMed  Google Scholar 

  • Zhang H, Compaore MK, Lee EG, Liao M, Zhang G, Sugimoto C, Fujisaki K, Nishikawa Y, Xuan X (2007) Apical membrane antigen 1 is a cross-reactive antigen between Neospora caninum and Toxoplasma gondii, and the anti-NcAMA1 antibody inhibits host cell invasion by both parasites. Mol Biochem Parasitol 151:205–212

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This study was supported by the “National Key Basic Research Program (973 program) of China” (2015CB150300), Beijing Municipal Natural Science Foundation (6131001), National Natural Science Foundation of China (31302075), and Modern Agroindustry Technology Research System (CARS-37).

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Authors and Affiliations

  1. Key Laboratory of Animal Epidemiology and Zoonosis, Ministry of Agriculture, and National Animal Protozoa Laboratory, College of Veterinary Medicine, China Agricultural University, Beijing, China

    Wensheng Li, Jing Liu, Jing Wang, Yong Fu, Huizhu Nan & Qun Liu

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  1. Wensheng Li
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  2. Jing Liu
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  3. Jing Wang
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  4. Yong Fu
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  5. Huizhu Nan
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  6. Qun Liu
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Correspondence to Qun Liu.

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Li, W., Liu, J., Wang, J. et al. Identification and characterization of a microneme protein (NcMIC6) in Neospora caninum . Parasitol Res 114, 2893–2902 (2015). https://doi.org/10.1007/s00436-015-4490-3

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  • DOI: https://doi.org/10.1007/s00436-015-4490-3

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