Skip to main content Accessibility help
×
Home
Hostname: page-component-8bbf57454-w5vlw Total loading time: 0.553 Render date: 2022-01-24T08:10:27.349Z Has data issue: true Feature Flags: { "shouldUseShareProductTool": true, "shouldUseHypothesis": true, "isUnsiloEnabled": true, "metricsAbstractViews": false, "figures": true, "newCiteModal": false, "newCitedByModal": true, "newEcommerce": true, "newUsageEvents": true }

Article contents

Molecular characterization of Neospora caninum MAG1, a dense granule protein secreted into the parasitophorous vacuole, and associated with the cyst wall and the cyst matrix

Published online by Cambridge University Press:  06 May 2010

CHRISTOPHE GUIONAUD
Affiliation:
Institute of Veterinary Pharmacology and Toxicology, Vetsuisse Faculty, University of Berne, Länggass-Strasse 124, CH-3012 Berne, Switzerland
ANDREW HEMPHILL*
Affiliation:
Institute of Parasitology, Vetsuisse Faculty, University of Berne, Länggass-Strasse 122, CH-3012 Berne, Switzerland
MEIKE MEVISSEN
Affiliation:
Institute of Veterinary Pharmacology and Toxicology, Vetsuisse Faculty, University of Berne, Länggass-Strasse 124, CH-3012 Berne, Switzerland
FERIAL ALAEDDINE*
Affiliation:
Institute of Veterinary Pharmacology and Toxicology, Vetsuisse Faculty, University of Berne, Länggass-Strasse 124, CH-3012 Berne, Switzerland
*
*Corresponding author: Institute of Veterinary Pharmacology and Toxicology, Vetsuisse Faculty, University of Berne, Länggass-Strasse 124, CH-3012 Berne, Switzerland. Tel: +41 31 631 22 41. Fax: +41 31 631 26 30. E-mail: ferial.alaeddine@vpi.unibe.ch

Summary

In Neospora caninum and Toxoplasma gondii, the parasitophorous vacuole (PV) is synthesized at the time of infection. During tachyzoite-to-bradyzoite stage conversion, the PV is later transformed into a tissue cyst that allows parasites to survive in their host for extended periods of time. We report on the characterization of NcMAG1, the N. caninum orthologue of T. gondii MAG1 (matrix antigen 1; TgMAG1). The 456 amino acid predicted NcMAG1 protein is 54% identical to TgMAG1. By immunoblotting, a rabbit antiserum raised against recombinant NcMAG1 detected a major product of ~67 kDa in extracts of N. caninum tachyzoite-infected Vero cells, which was stained more prominently in extracts of infected Vero cells treated to induce in vitro bradyzoite conversion. Immunofluorescence and TEM localized the protein mainly within the cyst wall and the cyst matrix. In both tachyzoites and bradyzoites, NcMAG1 was associated with the parasite dense granules. Comparison between NcMAG1 and TgMAG1 amino acid sequences revealed that the C-terminal conserved regions exhibit 66% identity, while the N-terminal variable regions exhibit only 32% identity. Antibodies against NcMAG1-conserved region cross-reacted with the orthologuous protein in T. gondii but those against the variable region did not. This indicates that the variable region possesses unique antigenic characteristics.

Type
Research Article
Copyright
Copyright © Cambridge University Press 2010

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

Abramoff, M. D., Magelhaes, P. J. and Ram, S. J. (2004). Image processing with Image J. Biophotonics International 11, 3642.Google Scholar
Adjogble, K. D., Mercier, C., Dubremetz, J. F., Hucke, C., Mackenzie, C. R., Cesbron-Delauw, M. F. and Daubener, W. (2004). GRA9, a new Toxoplasma gondii dense granule protein associated with the intravacuolar network of tubular membranes. International Journal for Parasitology 34, 12551264.CrossRefGoogle ScholarPubMed
Aguado-Martinez, A., Alvarez-Garcia, G., Fernandez-Garcia, A., Risco-Castillo, V., Marugan-Hernandez, V. and Ortega-Mora, L. M. (2009). Failure of a vaccine using immunogenic recombinant proteins rNcSAG4 and rNcGRA7 against neosporosis in mice. Vaccine 27, 73317338. doi: 10.1016/S0264-410X(09)01384-X.CrossRefGoogle ScholarPubMed
Ahn, H. J., Kim, S. and Nam, H. W. (2005). Host cell binding of GRA10, a novel, constitutively secreted dense granular protein from Toxoplasma gondii. Biochemical and Biophysical Research Communications 331, 614620.CrossRefGoogle ScholarPubMed
Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology 215, 403410.CrossRefGoogle ScholarPubMed
Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A. and Struhl, K. (1997). Current Protocols in Molecular Biology. John Wiley & Sons, Somerset, UK.Google Scholar
Barber, J. S., Holmdahl, O. J., Owen, M. R., Guy, F., Uggla, A. and Trees, A. J. (1995). Characterization of the first European isolate of Neospora caninum (Dubey, Carpenter, Speer, Topper and Uggla). Parasitology 111, 563568.CrossRefGoogle Scholar
Bendtsen, J. D., Nielsen, H., von Heijne, G. and Brunak, S. (2004). Improved prediction of signal peptides: SignalP 3.0. Journal of Molecular Biology 340, 783795.CrossRefGoogle ScholarPubMed
Beyer, T. V., Svezhova, N. V., Radchenko, A. I. and Sidorenko, N. V. (2002). Parasitophorous vacuole: morphofunctional diversity in different coccidian genera (a short insight into the problem). Cell Biology International 26, 861871.CrossRefGoogle Scholar
Bohne, W., Heesemann, J. and Gross, U. (1994). Reduced replication of Toxoplasma gondii is necessary for induction of bradyzoite-specific antigens: a possible role for nitric oxide in triggering stage conversion. Infection and Immunity 62, 17611767.Google ScholarPubMed
Bonfield, J. K., Smith, K. and Staden, R. (1995). A new DNA sequence assembly program. Nucleic Acids Research 23, 49924999.CrossRefGoogle ScholarPubMed
Chou, Q., Russell, M., Birch, D. E., Raymond, J. and Bloch, W. (1992). Prevention of pre-PCR mis-priming and primer dimerization improves low-copy-number amplifications. Nucleic Acids Research 20, 17171723.CrossRefGoogle ScholarPubMed
Contini, C., Cultrera, R., Seraceni, S., Segala, D., Romani, R., Fainardi, E., Cinque, P., Lazzarin, A. and Delia, S. (2002). The role of stage-specific oligonucleotide primers in providing effective laboratory support for the molecular diagnosis of reactivated Toxoplasma gondii encephalitis in patients with AIDS. Journal of Medical Microbiology 51, 879890.CrossRefGoogle ScholarPubMed
Coppens, I., Dunn, J. D., Romano, J. D., Pypaert, M., Zhang, H., Boothroyd, J. C. and Joiner, K. A. (2006). Toxoplasma gondii sequesters lysosomes from mammalian hosts in the vacuolar space. Cell 125, 261274.CrossRefGoogle ScholarPubMed
Debache, K., Guionaud, C., Alaeddine, F. and Hemphill, A. (2009). Intraperitoneal and intra-nasal vaccination of mice with three distinct recombinant Neospora caninum antigens results in differential effects with regard to protection against experimental challenge with Neospora caninum tachyzoites. Parasitology 137, 229240. doi: 10.1017/S0031182009991259.CrossRefGoogle ScholarPubMed
Di Cristina, M., Del Porto, P., Buffolano, W., Beghetto, E., Spadoni, A., Guglietta, S., Piccolella, E., Felici, F. and Gargano, N. (2004). The Toxoplasma gondii bradyzoite antigens BAG1 and MAG1 induce early humoral and cell-mediated immune responses upon human infection. Microbes and Infection 6, 164171.CrossRefGoogle ScholarPubMed
Dubey, J. P., Buxton, D. and Wouda, W. (2006). Pathogenesis of bovine neosporosis. Journal of Comparative Pathology 134, 267289.CrossRefGoogle ScholarPubMed
Dubey, J. P., Carpenter, J. L., Speer, C. A., Topper, M. J. and Uggla, A. (1988). Newly recognized fatal protozoan disease of dogs. Journal of the American Veterinary Medical Association 192, 12691285.Google ScholarPubMed
Edgar, R. C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Research 32, 17921797.CrossRefGoogle ScholarPubMed
Ferguson, D. J. (2004). Use of molecular and ultrastructural markers to evaluate stage conversion of Toxoplasma gondii in both the intermediate and definitive host. International Journal for Parasitology 34, 347360.CrossRefGoogle ScholarPubMed
Ferguson, D. J., and Parmley, S. F. (2002). Toxoplasma gondii MAG1 protein expression. Trends in Parasitology 18, 482.CrossRefGoogle ScholarPubMed
Fuchs, N., Sonda, S., Gottstein, B. and Hemphill, A. (1998). Differential expression of cell surface- and dense granule-associated Neospora caninum proteins in tachyzoites and bradyzoites. Journal of Parasitology 84, 753758.CrossRefGoogle ScholarPubMed
Gondim, L. F., McAllister, M. M., Pitt, W. C. and Zemlicka, D. E. (2004). Coyotes (Canis latrans) are definitive hosts of Neospora caninum. International Journal for Parasitology 34, 159161.CrossRefGoogle ScholarPubMed
Gross, U., Bormuth, H., Gaissmaier, C., Dittrich, C., Krenn, V., Bohne, W. and Ferguson, D. J. (1995). Monoclonal rat antibodies directed against Toxoplasma gondii suitable for studying tachyzoite-bradyzoite interconversion in vivo. Clinical and Diagnostic Laboratory Immunology 2, 542548.Google ScholarPubMed
Guo, Z. and Sherman, F. (1995). 3'-end-forming signals of yeast mRNA. Molecular and Cellular Biology 15, 59835990.CrossRefGoogle ScholarPubMed
Hemphill, A. and Gottstein, B. (1996). Identification of a major surface protein on Neospora caninum tachyzoites. Parasitology Research 82, 497504.CrossRefGoogle Scholar
Hemphill, A. and Croft, S. L. (1997). Electron microscopy of parasites. In Analytical Parasitology (ed. Rogan, M. and Graig, P.), pp. 227268. Springer Verlag, Heidelberg, Germany.CrossRefGoogle Scholar
Hemphill, A., Gottstein, B. and Kaufmann, H. (1996). Adhesion and invasion of bovine endothelial cells by Neospora caninum. Parasitology 112, 183197.CrossRefGoogle ScholarPubMed
Hemphill, A., Vonlaufen, N. and Naguleswaran, A. (2006). Cellular and immunological basis of the host-parasite relationship during infection with Neospora caninum. Parasitology 133, 261278. doi: 10.1017/S0031182006000485.CrossRefGoogle ScholarPubMed
Holec, L., Hiszczynska-Sawicka, E., Gasior, A., Brillowska-Dabrowska, A. and Kur, J. (2007). Use of MAG1 recombinant antigen for detection of Toxoplasma gondii infection in human. Clinical and Vaccine Immunology 14, 220225.CrossRefGoogle Scholar
Innes, E. A., Andrianarivo, A. G., Bjorkman, C., Williams, D. J. and Conrad, P. A. (2002). Immune responses to Neospora caninum and prospects for vaccination. Trends in Parasitology 18, 497504.CrossRefGoogle ScholarPubMed
Li, L., Crabtree, J., Fischer, S., Pinney, D., Stoeckert, C. J. Jr., Sibley, L. D. and Roos, D. S. (2004). ApiEST-DB: analyzing clustered EST data of the apicomplexan parasites. Nucleic Acids Research 32, D326–328.CrossRefGoogle ScholarPubMed
Li, L., Brunk, B. P., Kissinger, J. C., Pape, D., Tang, K., Cole, R. H., Martin, J., Wylie, T., Dante, M., Fogarty, S. J., Howe, D. K., Liberator, P., Diaz, C., Anderson, J., White, M., Jerome, M. E., Johnson, E. A., Radke, J. A., Stoeckert, C. J. Jr., Waterston, R. H., Clifton, S. W., Roos, D. S. and Sibley, L. D. (2003). Gene discovery in the apicomplexa as revealed by EST sequencing and assembly of a comparative gene database. Genome Research 13, 443454.CrossRefGoogle ScholarPubMed
Matrajt, M., Platt, C. D., Sagar, A. D., Lindsay, A., Moulton, C. and Roos, D. S. (2004). Transcript initiation, polyadenylation, and functional promoter mapping for the dihydrofolate reductase-thymidylate synthase gene of Toxoplasma gondii. Molecular and Biochemical Parasitology 137, 229238.CrossRefGoogle ScholarPubMed
Matz, M., Shagin, D., Bogdanova, E., Britanova, O., Lukyanov, S., Diatchenko, L. and Chenchik, A. (1999). Amplification of cDNA ends based on template-switching effect and step-out PCR. Nucleic Acids Research 27, 15581560.CrossRefGoogle ScholarPubMed
McAllister, M. M., Parmley, S. F., Weiss, L. M., Welch, V. J. and McGuire, A. M. (1996). An immunohistochemical method for detecting bradyzoite antigen (BAG5) in Toxoplasma gondii-infected tissues cross-reacts with a Neospora caninum bradyzoite antigen. Journal of Parasitology 82, 354355.CrossRefGoogle ScholarPubMed
McAllister, M. M., Dubey, J. P., Lindsay, D. S., Jolley, W. R., Wills, R. A. and McGuire, A. M. (1998). Dogs are definitive hosts of Neospora caninum. International Journal for Parasitology 28, 14731478.CrossRefGoogle ScholarPubMed
McGuire, A. M., McAllister, M. M., Jolley, W. R. and Anderson-Sprecher, R. C. (1997). A protocol for the production of Neospora caninum tissue cysts in mice. Journal of Parasitology 83, 647651.CrossRefGoogle ScholarPubMed
Melo, F. and Marti-Renom, M. A. (2006). Accuracy of sequence alignment and fold assessment using reduced amino acid alphabets. Proteins 63, 986995.CrossRefGoogle ScholarPubMed
Mercier, C., Adjogble, K. D., Daubener, W. and Delauw, M. F. (2005). Dense granules: are they key organelles to help understand the parasitophorous vacuole of all apicomplexa parasites? International Journal for Parasitology 35, 829849.CrossRefGoogle ScholarPubMed
Müller, N., Vonlaufen, N., Gianinazzi, C., Leib, S. L. and Hemphill, A. (2002). Application of real-time fluorescent PCR for quantitative assessment of Neospora caninum infections in organotypic slice cultures of rat central nervous system tissue. Journal of Clinical Microbiology 40, 252255.CrossRefGoogle ScholarPubMed
Nicholas, K. B., Nicholas, H. B. J. and Deerfield, D. W. I. (1997). GeneDoc: analysis and visualization of genetic variation. EMBNEW News 4.Google Scholar
Nielsen, H. V., Di Cristina, M., Beghetto, E., Spadoni, A., Petersen, E. and Gargano, N. (2006). Toxoplasma gondii: DNA vaccination with bradyzoite antigens induces protective immunity in mice against oral infection with parasite cysts. Experimental Parasitology 112, 274279.CrossRefGoogle ScholarPubMed
Parmley, S., Slifer, T. and Araujo, F. (2002). Protective effects of immunization with a recombinant cyst antigen in mouse models of infection with Toxoplasma gondii tissue cysts. Journal of Infectious Diseases 185 (Suppl 1), S90S95CrossRefGoogle ScholarPubMed
Parmley, S. F., Yang, S., Harth, G., Sibley, L. D., Sucharczuk, A. and Remington, J. S. (1994). Molecular characterization of a 65-kilodalton Toxoplasma gondii antigen expressed abundantly in the matrix of tissue cysts. Molecular and Biochemical Parasitology 66, 283296.CrossRefGoogle ScholarPubMed
Pfrepper, K. I., Enders, G., Gohl, M., Krczal, D., Hlobil, H., Wassenberg, D. and Soutschek, E. (2005). Seroreactivity to and avidity for recombinant antigens in toxoplasmosis. Clinical and Diagnostic Laboratory Immunology 12, 977982.Google ScholarPubMed
Reischl, U., Bretagne, S., Kruger, D., Ernault, P. and Costa, J. M. (2003). Comparison of two DNA targets for the diagnosis of Toxoplasmosis by real-time PCR using fluorescence resonance energy transfer hybridization probes. BMC Infectious Diseases 3, 7.CrossRefGoogle Scholar
Rettigner, C., Leclipteux, T., De Meerschman, F., Focant, C. and Losson, B. (2004). Survival, immune responses and tissue cyst production in outbred (Swiss white) and inbred (CBA/Ca) strains of mice experimentally infected with Neospora caninum tachyzoites. Veterinary Research 35, 225232.CrossRefGoogle ScholarPubMed
Risco-Castillo, V., Fernandez-Garcia, A., Zaballos, A., Aguado-Martinez, A., Hemphill, A., Rodriguez-Bertos, A., Alvarez-Garcia, G. and Ortega-Mora, L. M. (2007). Molecular characterisation of BSR4, a novel bradyzoite-specific gene from Neospora caninum. International Journal for Parasitology 37, 887896. doi: 10.1016/S0020-7519(07)00046-X.CrossRefGoogle ScholarPubMed
Robinson, P. A., Anderton, B. H. and Loviny, T. L. (1988). Nitrocellulose-bound antigen repeatedly used for the affinity purification of specific polyclonal antibodies for screening DNA expression libraries. Journal of Immunological Methods 108, 115122.CrossRefGoogle ScholarPubMed
Seeber, F. (1997). Consensus sequence of translational initiation sites from Toxoplasma gondii genes. Parasitology Research 83, 309311.CrossRefGoogle ScholarPubMed
Vonlaufen, N., Guetg, N., Naguleswaran, A., Müller, N., Bjorkman, C., Schares, G., von Blumroeder, D., Ellis, J. and Hemphill, A. (2004). In vitro induction of Neospora caninum bradyzoites in vero cells reveals differential antigen expression, localization, and host-cell recognition of tachyzoites and bradyzoites. Infection and Immunity 72, 576583.CrossRefGoogle ScholarPubMed
Vonlaufen, N., Müller, N., Keller, N., Naguleswaran, A., Bohne, W., McAllister, M. M., Bjorkman, C., Müller, E., Caldelari, R. and Hemphill, A. (2002). Exogenous nitric oxide triggers Neospora caninum tachyzoite-to-bradyzoite stage conversion in murine epidermal keratinocyte cell cultures. International Journal for Parasitology 32, 12531265.CrossRefGoogle ScholarPubMed
Weiss, L. M., Ma, Y. F., Takvorian, P. M., Tanowitz, H. B. and Wittner, M. (1998). Bradyzoite development in Toxoplasma gondii and the hsp70 stress response. Infection and Immunity 66, 32953302.Google ScholarPubMed
13
Cited by

Send article to Kindle

To send this article to your Kindle, first ensure no-reply@cambridge.org is added to your Approved Personal Document E-mail List under your Personal Document Settings on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part of your Kindle email address below. Find out more about sending to your Kindle. Find out more about sending to your Kindle.

Note you can select to send to either the @free.kindle.com or @kindle.com variations. ‘@free.kindle.com’ emails are free but can only be sent to your device when it is connected to wi-fi. ‘@kindle.com’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.

Find out more about the Kindle Personal Document Service.

Molecular characterization of Neospora caninum MAG1, a dense granule protein secreted into the parasitophorous vacuole, and associated with the cyst wall and the cyst matrix
Available formats
×

Send article to Dropbox

To send this article to your Dropbox account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Dropbox.

Molecular characterization of Neospora caninum MAG1, a dense granule protein secreted into the parasitophorous vacuole, and associated with the cyst wall and the cyst matrix
Available formats
×

Send article to Google Drive

To send this article to your Google Drive account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Google Drive.

Molecular characterization of Neospora caninum MAG1, a dense granule protein secreted into the parasitophorous vacuole, and associated with the cyst wall and the cyst matrix
Available formats
×
×

Reply to: Submit a response

Please enter your response.

Your details

Please enter a valid email address.

Conflicting interests

Do you have any conflicting interests? *