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Studying the Cell Biology of Apicomplexan Parasites Using Fluorescent Proteins

Published online by Cambridge University Press:  01 October 2004

Marc-Jan Gubbels  and
Boris Striepen
Marc-Jan Gubbels
Affiliation:
Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, GA 30602
Boris Striepen
Affiliation:
Center for Tropical and Emerging Global Diseases and Department of Cellular Biology, University of Georgia, Athens, GA 30602
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Abstract

The ability to transfect Apicomplexan parasites has revolutionized the study of this important group of pathogens. The function of specific genes can be explored by disruption of the locus or more subtly by introduction of altered or tagged versions. Using the transgenic reporter gene green fluorescent protein (GFP), cell biological processes can now be studied in living parasites and in real time. We review recent advances made using GFP-based experiments in the understanding of protein trafficking, organelle biogenesis, and cell division in Toxoplasma gondii and Plasmodium falciparum. A technical section provides a collection of basic experimental protocols for fluorescent protein expression in T. gondii. The combination of the in vivo marker GFP with an increasingly diverse genetic toolbox for T. gondii opens many exciting experimental opportunities, and emerging applications of GFP in genetic and pharmacological screens are discussed.

Keywords

ToxoplasmaPlasmodiumApicomplexaGFPgreen fluorescent proteinfluorescence microscopyin vivo microscopy
Type
Feature Articles
Information
Microscopy and Microanalysis , Volume 10 , Issue 5 , October 2004 , pp. 568 - 579
Copyright
© 2004 Microscopy Society of America

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References

REFERENCES

Adisa, A., Rug, M., Klonis, N., Foley, M., Cowman, A.F., & Tilley, L. (2003). The signal sequence of exported protein-1 directs the green fluorescent protein to the parasitophorous vacuole of transfected malaria parasites. J Biol Chem 278, 6532–6542.Google Scholar
Al-Anouti, F. & Ananvoranich, S. (2002). Comparative analysis of antisense RNA, double-stranded RNA, and delta ribozyme-mediated gene regulation in Toxoplasma gondii. Antisense Nucleic Acid Drug Dev 12, 275–281.Google Scholar
Black, M., Seeber, F., Soldati, D., Kim, K., & Boothroyd, J.C. (1995). Restriction enzyme-mediated integration elevates transformation frequency and enables co-transfection of Toxoplasma gondii. Mol Biochem Parasitol 74, 55–63.Google Scholar
Black, M.W., Arrizabalaga, G., & Boothroyd, J.C. (2000). Ionophore-resistant mutants of Toxoplasma gondii reveal host cell permeabilization as an early event in egress. Mol Cell Biol 20, 9399–9408.Google Scholar
Boothroyd, J.C., Kim, K., Pfefferkorn, E.R., Sibley, D.L., & Soldati, D. (1994). Toxoplasma gondii as a paradigm for the use of gentics in the study of protozoan parasites. Methods Mol Genet 6, 1–29.Google Scholar
Bradley, P.J. & Boothroyd, J.C. (2001). The pro-region of Toxoplasma ROP1 is a rhoptry-targeting signal. Int J Parasitol 31, 1177–1186.Google Scholar
Carey, K.L., Westwood, N.J., Mitchison, T.J., & Ward, G.E. (2004). A small-molecule approach to studying invasive mechanisms of Toxoplasma gondii. Proc Natl Acad Sci USA 101, 7433–7438.Google Scholar
Cerede, O., Dubremetz, J.F., Bout, D., & Lebrun, M. (2002). The Toxoplasma gondii protein MIC3 requires pro-peptide cleavage and dimerization to function as adhesin. EMBO J 21, 2526–2536.Google Scholar
Chalfie, M., Tu, Y., Euskirchen, G., Ward, W.W., & Prasher, D.C. (1994). Green fluorescent protein as a marker for gene expression. Science 263, 802–805.Google Scholar
Crabb, B.S., Cooke, B.M., Reeder, J.C., Waller, R.F., Caruana, S.R., Davern, K.M., Wickham, M.E., Brown, G.V., Coppel, R.L., & Cowman, A.F. (1997). Targeted gene disruption shows that knobs enable malaria-infected red cells to cytoadhere under physiological shear stress. Cell 89, 287–296.Google Scholar
de Koning-Ward, T.F., Thomas, A.W., Waters, A.P., & Janse, C.J. (1998). Stable expression of green fluorescent protein in blood and mosquito stages of Plasmodium berghei. Mol Biochem Parasitol 97, 247–252.Google Scholar
DeRocher, A., Hagen, C.B., Froehlich, J.E., Feagin, J.E., & Parsons, M. (2000). Analysis of targeting sequences demonstrates that trafficking to the Toxoplasma gondii plastid branches off the secretory system. J Cell Sci 113, 3969–3977.Google Scholar
Dobrowolski, J.M. & Sibley, L.D. (1996). Toxoplasma invasion of mammalian cells is powered by the actin cytoskeleton of the parasite. Cell 84, 933–939.Google Scholar
Donald, R.G., Carter, D., Ullman, B., & Roos, D.S. (1996). Insertional tagging, cloning, and expression of the Toxoplasma gondii hypoxanthine-xanthine-guanine phosphoribosyltransferase gene. Use as a selectable marker for stable transformation. J Biol Chem 271, 14010–14019.Google Scholar
Donald, R.G. & Roos, D.S. (1993). Stable molecular transformation of Toxoplasma gondii: A selectable dihydrofolate reductase-thymidylate synthase marker based on drug-resistance mutations in malaria. Proc Natl Acad Sci USA 90, 11703–11707.Google Scholar
Fichera, M.E. & Roos, D.S. (1997). A plastid organelle as a drug target in apicomplexan parasites. Nature 390, 407–409.Google Scholar
Foth, B.J., Ralph, S.A., Tonkin, C.J., Struck, N.S., Fraunholz, M., Roos, D.S., Cowman, A.F., & McFadden, G.I. (2003). Dissecting apicoplast targeting in the malaria parasite Plasmodium falciparum. Science 299, 705–708.Google Scholar
Gardner, M.J., Hall, N., Fung, E., White, O., Berriman, M., Hyman, R.W., Carlton, J.M., Pain, A., Nelson, K.E., Bowman, S., Paulsen, I.T., James, K., Eisen, J.A., Rutherford, K., Salzberg, S.L., Craig, A., Kyes, S., Chan, M.S., Nene, V., Shallom, S.J., Suh, B., Peterson, J., Angiuoli, S., Pertea, M., Allen, J., Selengut, J., Haft, D., Mather, M.W., Vaidya, A.B., Martin, D.M., Fairlamb, A.H., Fraunholz, M.J., Roos, D.S., Ralph, S.A., McFadden, G.I., Cummings, L.M., Subramanian, G.M., Mungall, C., Venter, J.C., Carucci, D.J., Hoffman, S.L., Newbold, C., Davis, R.W., Fraser, C.M., & Barrell, B. (2002). Genome sequence of the human malaria parasite Plasmodium falciparum. Nature 419, 498–511.Google Scholar
Gubbels, M.-J., Li, C., & Striepen, B. (2003). High throughput growth assay for Toxoplasma gondii using yellow fluorescent protein. Antimicrob Agents Chemother 47, 309–316.Google Scholar
Gubbels, M.J., Wieffer, M., & Striepen, B. (2004). Fluorescent protein tagging in Toxoplasma gondii: Identification of a novel inner membrane complex component conserved among Apicomplexa. Mol Biochem Parasitol (in press).Google Scholar
Hager, K.M., Striepen, B., Tilney, L.G., & Roos, D.S. (1999). The nuclear envelope serves as an intermediary between the ER and Golgi complex in the intracellular parasite Toxoplasma gondii. J Cell Sci 112, 2631–2638.Google Scholar
Hartley, J.L., Temple, G.F., & Brasch, M.A. (2000). DNA cloning using in vitro site-specific recombination. Genome Res 10, 1788–1795.Google Scholar
He, C.Y., Shaw, M.K., Pletcher, C.H., Striepen, B., Tilney, L.G., & Roos, D.S. (2001a). A plastid segregation defect in the protozoan parasite Toxoplasma gondii. EMBO J 20, 330–339.Google Scholar
He, C.Y., Striepen, B., Pletcher, C.H., Murray, J.M., & Roos, D.S. (2001b). Targeting and processing of nuclear-encoded apicoplast proteins in plastid segregation mutants of Toxoplasma gondii. J Biol Chem 276, 28436–28442.Google Scholar
Hettmann, C., Herm, A., Geiter, A., Frank, B., Schwarz, E., Soldati, T., & Soldati, D. (2000). A dibasic motif in the tail of a class XIV apicomplexan myosin is an essential determinant of plasma membrane localization. Mol Biol Cell 11, 1385–1400.Google Scholar
Hu, K., Mann, T., Striepen, B., Beckers, C.J., Roos, D.S., & Murray, J.M. (2002a). Daughter cell assembly in the protozoan parasite Toxoplasma gondii. Mol Biol Cell 13, 593–606.Google Scholar
Hu, K., Roos, D.S., & Murray, J.M. (2002b). A novel polymer of tubulin forms the conoid of Toxoplasma gondii. J Cell Biol 156, 1039–1050.Google Scholar
Joiner, K.A. & Roos, D.S. (2002). Secretory traffic in the eukaryotic parasite Toxoplasma gondii: Less is more. J Cell Biol 157, 557–563.Google Scholar
Kadekoppala, M., Kline, K., Akompong, T., & Haldar, K. (2000). Stable expression of a new chimeric fluorescent reporter in the human malaria parasite Plasmodium falciparum. Infect Immun 68, 2328–2332.Google Scholar
Karsten, V., Qi, H., Beckers, C.J., Reddy, A., Dubremetz, J.F., Webster, P., & Joiner, K.A. (1998). The protozoan parasite Toxoplasma gondii targets proteins to dense granules and the vacuolar space using both conserved and unusual mechanisms. J Cell Biol 141, 1323–1333.Google Scholar
Kim, K., Eaton, M.S., Schubert, W., Wu, S., & Tang, J. (2001). Optimized expression of green fluorescent protein in Toxoplasma gondii using thermostable green fluorescent protein mutants. Mol Biochem Parasitol 113, 309–313.Google Scholar
Kim, K., Soldati, D., & Boothroyd, J.C. (1993). Gene replacement in Toxoplasma gondii with chloramphenicol acetyltransferase as selectable marker. Science 262, 911–914.Google Scholar
Mann, T., Gaskins, E., & Beckers, C. (2002). Proteolytic processing of TgIMC1 during maturation of the membrane skeleton of Toxoplasma gondii. J Biol Chem 277, 41240–41246.Google Scholar
Matrajt, M., Nishi, M., Fraunholz, M.J., Peter, O., & Roos, D.S. (2002). Amino-terminal control of transgenic protein expression levels in Toxoplasma gondii. Mol Biochem Parasitol 120, 285–289.Google Scholar
McIntosh, M.T., Drozdowicz, Y.M., Laroiya, K., Rea, P.A., & Vaidya, A.B. (2001). Two classes of plant-like vacuolar-type H(+)-pyrophosphatases in malaria parasites. Mol Biochem Parasitol 114, 183–195.Google Scholar
Meissner, M., Reiss, M., Viebig, N., Carruthers, V.B., Toursel, C., Tomavo, S., Ajioka, J.W., & Soldati, D. (2002a). A family of transmembrane microneme proteins of Toxoplasma gondii contain EGF-like domains and function as escorters. J Cell Sci 115, 563–574.Google Scholar
Meissner, M., Schluter, D., & Soldati, D. (2002b). Role of Toxoplasma gondii myosin A in powering parasite gliding and host cell invasion. Science 298, 837–840.Google Scholar
Morrissette, N.S. & Sibley, L.D. (2002). Disruption of microtubules uncouples budding and nuclear division in Toxoplasma gondii. J Cell Sci 115, 1017–1025.Google Scholar
Mota, M.M., Thathy, V., Nussenzweig, R.S., & Nussenzweig, V. (2001). Gene targeting in the rodent malaria parasite Plasmodium yoelii. Mol Biochem Parasitol 113, 271–278.Google Scholar
Ormo, M., Cubitt, A.B., Kallio, K., Gross, L.A., Tsien, R.Y., & Remington, S.J. (1996). Crystal structure of the Aequorea victoria green fluorescent protein. Science 273, 1392–1395.Google Scholar
Osteryoung, K.W. (2001). Organelle fission in eukaryotes. Curr Opin Microbiol 4, 639–646.Google Scholar
Pelletier, L., Stern, C.A., Pypaert, M., Sheff, D., Ngo, H.M., Roper, N., He, C.Y., Hu, K., Toomre, D., Coppens, I., Roos, D.S., Joiner, K.A., & Warren, G. (2002). Golgi biogenesis in Toxoplasma gondii. Nature 418, 548–552.Google Scholar
Pfefferkorn, E.R. & Pfefferkorn, L.C. (1976). Toxoplasma gondii: Isolation and preliminary characterization of temperature-sensitive mutants. Exp Parasitol 39, 365–376.Google Scholar
Radke, J.R., Guerini, M.N., & White, M.W. (2000). Toxoplasma gondii: Characterization of temperature-sensitive tachyzoite cell cycle mutants. Exp Parasitol 96, 168–177.Google Scholar
Radke, J.R., Striepen, B., Guerini, M.N., Jerome, M.E., Roos, D.S., & White, M.W. (2001). Defining the cell cycle for the tachyzoite stage of Toxoplasma gondii. Mol Biochem Parasitol 115, 165–175.Google Scholar
Reiss, M., Viebig, N., Brecht, S., Fourmaux, M.N., Soete, M., Di Cristina, M., Dubremetz, J.F., & Soldati, D. (2001). Identification and characterization of an escorter for two secretory adhesins in Toxoplasma gondii. J Cell Biol 152, 563–578.Google Scholar
Roos, D.S., Crawford, M.J., Donald, R.G., Fraunholz, M., Harb, O.S., He, C.Y., Kissinger, J.C., Shaw, M.K., & Striepen, B. (2002). Mining the Plasmodium genome database to define organellar function: What does the apicoplast do? Philos Trans R Soc Lond B Biol Sci 357, 35–46.Google Scholar
Roos, D.S., Crawford, M.J., Donald, R.G., Kissinger, J.C., Klimczak, L.J., & Striepen, B. (1999). Origin, targeting, and function of the apicomplexan plastid. Curr Opin Microbiol 2, 426–432.Google Scholar
Roos, D.S., Sullivan, W.J., Striepen, B., Bohne, W., & Donald, R.G. (1997). Tagging genes and trapping promoters in Toxoplasma gondii by insertional mutagenesis. Methods 13, 112–122.Google Scholar
Sheffield, H.G. & Melton, M.L. (1968). The fine structure and reproduction of Toxoplasma gondii. J Parasitol 54, 209–226.Google Scholar
Shorter, J. & Warren, G. (2002). Golgi architecture and inheritance. Annu Rev Cell Dev Biol 18, 379–420.Google Scholar
Singh, U., Brewer, J.L., & Boothroyd, J.C. (2002). Genetic analysis of tachyzoite to bradyzoite differentiation mutants in Toxoplasma gondii reveals a hierarchy of gene induction. Mol Microbiol 44, 721–733.Google Scholar
Striepen, B., Crawford, M.J., Shaw, M.K., Tilney, L.G., Seeber, F., & Roos, D.S. (2000). The plastid of Toxoplasma gondii is divided by association with the centrosomes. J Cell Biol 151, 1423–1434.Google Scholar
Striepen, B., He, C.Y., Matrajt, M., Soldati, D., & Roos, D.S. (1998). Expression, selection, and organellar targeting of the green fluorescent protein in Toxoplasma gondii. Mol Biochem Parasitol 92, 325–338.Google Scholar
Striepen, B., Soldati, D., Garcia-Reguet, N., Dubremetz, J.F., & Roos, D.S. (2001). Targeting of soluble proteins to the rhoptries and micronemes in Toxoplasma gondii. Mol Biochem Parasitol 113, 45–53.Google Scholar
Striepen, B., White, M.W., Li, C., Guerini, M.N., Malik, S.B., Logsdon, J.M., Jr., Liu, C., & Abrahamsen, M.S. (2002). Genetic complementation in apicomplexan parasites. Proc Natl Acad Sci USA 99, 6304–6309.Google Scholar
Sultan, A.A., Thathy, V., Nussenzweig, V., & Menard, R. (1999). Green fluorescent protein as a marker in Plasmodium berghei transformation. Infect Immun 67, 2602–2606.Google Scholar
Swedlow, J.R., Hu, K., Andrews, P.D., Roos, D.S., & Murray, J.M. (2002). Measuring tubulin content in Toxoplasma gondii: A comparison of laser-scanning confocal and wide-field fluorescence microscopy. Proc Natl Acad Sci USA 99, 2014–2019.Google Scholar
Uyetake, L., Ortega-Barria, E., & Boothroyd, J.C. (2001). Isolation and characterization of a cold-sensitive attachment/invasion mutant of Toxoplasma gondii. Exp Parasitol 97, 55–59.Google Scholar
VanWye, J.D. & Haldar, K. (1997). Expression of green fluorescent protein in Plasmodium falciparum. Mol Biochem Parasitol 87, 225–229.Google Scholar
Waller, R.F., Keeling, P.J., Donald, R.G., Striepen, B., Handman, E., Lang-Unnasch, N., Cowman, A.F., Besra, G.S., Roos, D.S., & McFadden, G.I. (1998). Nuclear-encoded proteins target to the plastid in Toxoplasma gondii and Plasmodium falciparum. Proc Natl Acad Sci USA 95, 12352–12357.Google Scholar
Waller, R.F., Reed, M.B., Cowman, A.F., & McFadden, G.I. (2000). Protein trafficking to the plastid of Plasmodium falciparum is via the secretory pathway. EMBO J 19, 1794–1802.Google Scholar
Ward, G.E., Carey, K.L., & Westwood, N.J. (2002). Using small molecules to study big questions in cellular microbiology. Cell Microbiol 4, 471–482.Google Scholar
Ward, T.H., Polishchuk, R.S., Caplan, S., Hirschberg, K., & Lippincott-Schwartz, J. (2001). Maintenance of Golgi structure and function depends on the integrity of ER export. J Cell Biol 155, 557–570.Google Scholar
Wickham, M.E., Rug, M., Ralph, S.A., Klonis, N., McFadden, G.I., Tilley, L., & Cowman, A.F. (2001). Trafficking and assembly of the cytoadherence complex in Plasmodium falciparum-infected human erythrocytes. EMBO J 20, 5636–5649.Google Scholar
Wilson, R.J. (2002). Progress with parasite plastids. J Mol Biol 319, 257–274.Google Scholar