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Visualization of Toxoplasma gondii stage conversion by expression of stage-specific dual fluorescent proteins

Published online by Cambridge University Press:  16 April 2009

A. UNNO ,
K. SUZUKI ,
T. BATANOVA ,
S.-Y. CHA ,
H.-K. JANG ,
K. KITOH  and
Y. TAKASHIMA
A. UNNO
Affiliation:
Department of Veterinary Parasitological Diseases, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan
K. SUZUKI
Affiliation:
Laboratory of Veterinary Epizootiology, Nihon University, Kameino 1866, Fujisawa 252-8510, Japan
T. BATANOVA
Affiliation:
Department of Veterinary Parasitological Diseases, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan Research Center of Virology and Biotechnology, Vector, Koltsovo, Novosibirsk region, 630559, Russia
S.-Y. CHA
Affiliation:
Department of Veterinary Parasitological Diseases, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan Department of Infectious Diseases and Avian Diseases, College of Veterinary Medicine, Chonbuk National University, Duckjin-Dong 664-14, Duckjin-Ku, Jeonju 561-756, Korea
H.-K. JANG
Affiliation:
Department of Infectious Diseases and Avian Diseases, College of Veterinary Medicine, Chonbuk National University, Duckjin-Dong 664-14, Duckjin-Ku, Jeonju 561-756, Korea
K. KITOH
Affiliation:
Department of Veterinary Parasitological Diseases, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan
Y. TAKASHIMA*
Affiliation:
Department of Veterinary Parasitological Diseases, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan
*
*Corresponding author: Department of Veterinary Parasitological Diseases, Gifu University, Yanagido 1-1, Gifu 501-1193, Japan. Tel: and Fax: +81 58 293 2956. E-mail: atakashi@gifu-u.ac.jp
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Summary

To recognize the stage conversion of Toxoplasma gondii between tachyzoite and bradyzoite in live host cells, a transgenic T. gondii line, which expressed stage-specific red and green fluorescence, was constructed. T. gondii PLK strain tachyzoites were stably transformed with genes encoding red fluorescent protein (DsRed Express) and green fluorescent protein (GFP) under the control of tachyzoite-specific SAG1 and bradyzoite-specific BAG1 promoters, respectively. The resulting transgenic parasite was designated PLK/DUAL. When PLK/DUAL was cultured in pH 7·0 medium, the PLK/DUAL zoites expressed red fluorescence, but no detectable levels of green fluorescence were observed. The PLK/DUAL zoites reacted with anti-SAG1 antibody, but not anti-BAG1 antiserum. When PLK/DUAL was cultured under high pH conditions, or in the presence of the p38 MAPK inhibitor SB202190, a small number of zoites expressed green fluorescence and were BAG1 positive. C57BL/6J mice were infected with PLK/DUAL tachyzoites. During the acute and reactivating phase, zoites expressed red fluorescence. However, green fluorescence was not detectable. By contrast, latent cysts expressed green fluorescence. The stage-specific dual fluorescence of PLK/DUAL facilitates identification of the parasitic stage in live cells, with the advantage that fixation or immunostaining is not required.

Keywords

Toxoplasma gondiistage conversiondual fluorescent proteins
Type
Research Article
Information
Parasitology , Volume 136 , Issue 6 , May 2009 , pp. 579 - 588
Copyright
Copyright © Cambridge University Press 2009

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References

REFERENCES

Bohne, W., Heesemann, J. and Gross, U. (1993). Coexistence of heterogeneous populations of Toxoplasma gondii parasites within parasitophorous vacuoles of murine macrophages as revealed by a bradyzoite-specific monoclonal antibody. Parasitology Research 79, 485–487. doi:10.1007/BF00931588.CrossRefGoogle ScholarPubMed
Bohne, W., Heesemann, J. and Gross, U. (1993). Induction of bradyzoite-specific Toxoplasma gondii antigens in gamma interferon-treated mouse macrophages. Infection and Immunity 61, 1141–1145.CrossRefGoogle ScholarPubMed
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, 1761–1767.CrossRefGoogle ScholarPubMed
Bohne, W., Wirsing, A. and Gross, U. (1997). Bradyzoite-specific gene expression in Toxoplasma gondii requires minimal genomic elements. Molecular and Biochemical Parasitology 85, 89–98. doi: 10.1016/S0166-6851(96)02814-9.CrossRefGoogle ScholarPubMed
Bohne, W., Hunter, C. A., White, M. W., Ferguson, D. J., Gross, U. and Roos, D. S. (1998). Targeted disruption of the bradyzoite-specific gene BAG1 does not prevent tissue cyst formation in Toxoplasma gondii. Molecular and Biochemical Parasitology 92, 291–301. doi:10.1016/S0166-6851(97)00236-3.CrossRefGoogle Scholar
Carruthers, V. B. and Suzuki, Y. (2007). Effects of Toxoplasma gondii infection on the brain. Schizophrenia Bulletin 33, 745–751. doi:10.1093/schbul/sbm008.CrossRefGoogle ScholarPubMed
Cleary, M. D., Singh, U., Blader, I. J., Brewer, J. L. and Boothroyd, J. C. (2002). Toxoplasma gondii asexual development: identification of developmentally regulated genes and distinct patterns of gene expression. Eukaryotic Cell 1, 329–340. doi:10.1128/EC.1.3.329-340.2002.CrossRefGoogle ScholarPubMed
Day, R. N. and Schaufele, F. (2008). Fluorescent protein tools for studying protein dynamics in living cells: a review. Journal of Biomedical Optics 13, 031202. doi:10.1117/12.805530.CrossRefGoogle ScholarPubMed
Dubey, J. P. (1987). Toxoplasma gondii cysts in placentas of experimentally infected sheep. American Journal of Veterinary Research 48, 352–353.Google 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, 347–360. doi:10.1016/j.ijpara.2003.11.024.CrossRefGoogle ScholarPubMed
Ferguson, D. J., Hutchison, W. M. and Pettersen, E. (1989). Tissue cyst rupture in mice chronically infected with Toxoplasma gondii. An immunocytochemical and ultrastructural study. Parasitology Research 75, 599–603. doi:10.1007/BF00930955.CrossRefGoogle ScholarPubMed
Ferreira da Silva Mda, F., Barbosa, H. S., Gross, U. and Lüder, C. G. (2008). Stress-related and spontaneous stage differentiation of Toxoplasma gondii. Molecular Biosystems 4, 824–834. doi:10.1039/b800520f.CrossRefGoogle ScholarPubMed
Fouts, A. E. and Boothroyd, J. C. (2007). Infection with Toxoplasma gondii bradyzoites has a diminished impact on host transcript levels relative to tachyzoite infection. Infection and Immunity 75, 634–642. doi: 10.1128/IAI.01228-06.CrossRefGoogle Scholar
Grigg, M. E., Bonnefoy, S., Hehl, A. B., Suzuki, Y. and Boothroyd, J. C. (2001). Success and virulence in Toxoplasma as the result of sexual recombination between two distinct ancestries. Science 294, 161–165. doi: 10.1126/science.1061888.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, 542–548.CrossRefGoogle ScholarPubMed
Gross, U., Kempf, M. C., Seeber, F., Lüder, C. G., Lugert, R. and Bohne, W. (1997). Reactivation of chronic toxoplasmosis: is there a link to strain-specific differences in the parasite? Behring Institute Mitteilungen 99, 97–106.Google Scholar
Innes, E. A. (1997). Toxoplasmosis: comparative species susceptibility and host immune response. Comparative Immunology, Microbiology and Infectious Disease 20, 131–138. doi:10.1016/S0147-9571(96)00038-0.CrossRefGoogle ScholarPubMed
Lüder, C. G., Giraldo-Velásquez, M., Sendtner, M. and Gross, U. (1999). Toxoplasma gondii in primary rat CNS cells: differential contribution of neurons, astrocytes, and microglial cells for the intracerebral development and stage differentiation. Experimental Parasitology 93, 23–32. doi:10.1006/expr.1999.4421.CrossRefGoogle ScholarPubMed
Lyons, R. E., Lyons, K., McLeod, R. and Roberts, C. W. (2001). Construction and validation of a polycompetitor construct (SWITCH) for use in competitive RT-PCR to assess tachyzoite-bradyzoite interconversion in Toxoplasma gondii. Parasitology 123, 433–439. doi: 10.1017/S003118200100868X.CrossRefGoogle ScholarPubMed
Lyons, R. E., McLeod, R. and Roberts, C. W. (2002). Toxoplasma gondii tachyzoite-bradyzoite interconversion. Trends in Parasitology 18, 198–201. doi:10.1016/S1471-4922(02)02248-1.CrossRefGoogle ScholarPubMed
Montoya, J. G. and Liesenfeld, O. (2004). Toxoplasmosis. The Lancet 363, 1965–1976. doi:10.1016/S0140-6736(04)16412-X.CrossRefGoogle ScholarPubMed
Nishikawa, Y., Zhang, H., Ibrahim, H. M., Ui, F., Ogiso, A. and Xuan, X. (2008). Construction of Toxoplasma gondii bradyzoite expressing the green fluorescent protein. Parasitology International 57, 219–222. doi:10.1016/j.parint.2007.10.004.CrossRefGoogle ScholarPubMed
Niwa, H., Yamamura, K. and Miyazaki, J. (1991). Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene 108, 193–199. doi:10.1016/0378-1119(91)90434-D.Google ScholarPubMed
Pfefferkorn, E. R. and Borotz, S. E. (1994). Toxoplasma gondii: characterization of a mutant resistant to 6-thioxanthine. Experimental Parasitology 79, 374–382. doi:10.1006/expr.1994.1099.CrossRefGoogle ScholarPubMed
Radke, J. R., Donald, R. G., Eibs, A., Jerome, M. E., Behnke, M. S., Liberator, P. and White, M. W. (2006). Changes in the expression of human cell division autoantigen-1 influence Toxoplasma gondii growth and development. PLoS Pathogens 2, e105. doi:10.1371/journal.ppat.0020105.CrossRefGoogle ScholarPubMed
Roos, D. S., Donald, R. G., Morrissette, N. S. and Moulton, A. L. (1994). Molecular tools for genetic dissection of the protozoan parasite Toxoplasma gondii. Methods in Cell Biology 45, 27–63. doi:10.1016/S0091-679X(08)61845-2.CrossRefGoogle ScholarPubMed
Saeij, J. P., Coller, S., Boyle, J. P., Jerome, M. E., White, M. W. and Boothroyd, J. C. (2007). Toxoplasma co-opts host gene expression by injection of a polymorphic kinase homologue. Nature 445, 324–327. doi:10.1038/nature05395.CrossRefGoogle ScholarPubMed
Shiono, Y., Mun, H. S., He, N., Nakazaki, Y., Fang, H., Furuya, M., Aosai, F. and Yano, A. (2007). Maternal-fetal transmission of Toxoplasma gondii in interferon-gamma deficient pregnant mice. Parasitology International 56, 141–148. doi:10.1016/j.parint.2007.01.008.CrossRefGoogle ScholarPubMed
Silva, N. M., Gazzinelli, R. T., Silva, D. A., Ferro, E. A., Kasper, L. H. and Mineo, J. R. (1998). Expression of Toxoplasma gondii-specific heat shock protein 70 during in vivo conversion of bradyzoites to tachyzoites. Infection and Immunity 66, 3959–3963.CrossRefGoogle ScholarPubMed
Soete, M., Fortier, B., Camus, D. and Dubremetz, J. F. (1993). Toxoplasma gondii: kinetics of bradyzoite–tachyzoite interconversion in vitro. Experimental Parasitology 76, 259–264. doi:10.1006/expr.1993.1031.CrossRefGoogle ScholarPubMed
Soldati, D. and Boothroyd, J. C. (1993). Transient transfection and expression in the obligate intracellular parasite Toxoplasma gondii. Science 260, 349–352. doi: 10.1126/science.8469986.CrossRefGoogle ScholarPubMed
Sugimoto, K., Uema, M., Sagara, H., Tanaka, M., Sata, T., Hashimoto, Y. and Kawaguchi, Y. (2008). Simultaneous tracking of capsid, tegument, and envelope protein localization in living cells infected with triply fluorescent herpes simplex virus 1. Journal of Virology 82, 5198–5211. doi:10.1128/JVI.02681-07.CrossRefGoogle ScholarPubMed
Takashima, Y., Suzuki, K., Xuan, X., Nishikawa, Y., Unno, A. and Kitoh, K. (2008). Detection of the initial site of Toxoplasma gondii reactivation in brain tissue. International Journal for Parasitology 38, 601–607. doi:10.1016/j.ijpara.2007.09.017.CrossRefGoogle ScholarPubMed
Turner, G. V. (1978). Some aspects of the pathogenesis and comparative pathology of toxoplasmosis. Journal of the South African Veterinary Association 49, 3–8.Google ScholarPubMed
Unno, A., Suzuki, K., Xuan, X., Nishikawa, Y., Kitoh, K. and Takashima, Y. (2008). Dissemination of extracellular and intracellular Toxoplasma gondii tachyzoites in the blood flow. Parasitology International 57, 515–8. doi:10.1016/j.parint.2008.06.004.CrossRefGoogle ScholarPubMed
Vanchinathan, P., Brewer, J. L., Harb, O. S., Boothroyd, J. C. and Singh, U. (2005). Disruption of a locus encoding a nucleolar zinc finger protein decreases tachyzoite-to-bradyzoite differentiation in Toxoplasma gondii. Infection and Immunity 73, 6680–6688. doi: 10.1128/IAI.73.10.6680-6688.2005.CrossRefGoogle ScholarPubMed
Zhang, G., Huong, V. T., Battur, B., Zhou, J., Zhang, H., Liao, M., Kawase, O., Lee, E. G., Dautu, G., Igarashi, M., Nishikawa, Y. and Xuan, X. (2007). A heterologous prime-boost vaccination regime using DNA and a vaccinia virus, both expressing GRA4, induced protective immunity against Toxoplasma gondii infection in mice. Parasitology 134, 1339–1346. doi: 10.1017/S0031182007002892.CrossRefGoogle Scholar