High-Throughput Growth Assay for Toxoplasma gondii Using Yellow Fluorescent Protein

ABSTRACT A high-throughput growth assay for the protozoan parasite Toxoplasma gondii was developed based on a highly fluorescent transgenic parasite line. These parasites are stably transfected with a tandem yellow fluorescent protein (YFP) and are 1,000 times more fluorescent than the wild type. Parasites were inoculated in optical-bottom 384-well culture plates containing a confluent monolayer of host cells, and growth was monitored by using a fluorescence plate reader. The signal was linearly correlated with parasite numbers over a wide array. Direct comparison of the YFP growth assay with the β-galactosidase growth assay by using parasites expressing both reporters demonstrated that the assays' sensitivities were comparable but that the accuracy of the YFP assay was higher, especially at higher numbers of parasites per well. Determination of the 50%-inhibitory concentrations of three known growth-inhibiting drugs (cytochalasin D, pyrimethamine, and clindamycin) resulted in values comparable to published data. The delayed parasite death kinetics of clindamycin could be measured without modification of the assay, making this assay very versatile. Additionally, the temperature-dependent effect of pyrimethamine was assayed in both wild-type and engineered drug-resistant parasites. Lastly, the development of mycophenolic acid resistance after transfection of a resistance gene in T. gondii was followed. In conclusion, the YFP growth assay limits pipetting steps to a minimum, is highly versatile and amendable to automation, and should enable rapid screening of compounds to fulfill the need for more efficient and less toxic antiparasitic drugs.

[1]  Richard N. Day,et al.  Fluorescent protein spectra. , 2001, Journal of cell science.

[2]  D. Roos,et al.  The Plastid of Toxoplasma gondii Is Divided by Association with the Centrosomes , 2000, The Journal of cell biology.

[3]  E. Pfefferkorn,et al.  Specific labeling of intracellular Toxoplasma gondii with uracil. , 1977, The Journal of protozoology.

[4]  M. Chalfie.  GREEN FLUORESCENT PROTEIN , 1995, Photochemistry and photobiology.

[5]  J. Couvreur,et al.  Congenital toxoplasmosis. A prospective study of 378 pregnancies. , 1974, The New England journal of medicine.

[6]  D. Roos,et al.  Daughter cell assembly in the protozoan parasite Toxoplasma gondii. , 2002, Molecular biology of the cell.

[7]  A. Hehl,et al.  Expression of Green Fluorescent Protein as a Marker for Effects of Antileishmanial Compounds In Vitro , 2001, Antimicrobial Agents and Chemotherapy.

[8]  J. Boothroyd,et al.  Transient transfection and expression in the obligate intracellular parasite Toxoplasma gondii , 1993, Science.

[9]  P. Hugo,et al.  Detection of a decrease in green fluorescent protein fluorescence for the monitoring of cell death: an assay amenable to high-throughput screening technologies. , 2001, Cytometry.

[10]  W E Fahl,et al.  Development of a green fluorescent protein microplate assay for the screening of chemopreventive agents. , 2000, Analytical biochemistry.

[11]  David S. Roos,et al.  A plastid organelle as a drug target in apicomplexan parasites , 1997, Nature.

[12]  P. Winstanley,et al.  Modern chemotherapeutic options for malaria. , 2001, The Lancet. Infectious diseases.

[13]  J. Boothroyd,et al.  Use of Toxoplasma gondii expressing beta-galactosidase for colorimetric assessment of drug activity in vitro , 1997, Antimicrobial agents and chemotherapy.

[14]  David S. Roos,et al.  A novel polymer of tubulin forms the conoid of Toxoplasma gondii , 2002, The Journal of cell biology.

[15]  S. Sattar,et al.  Rapid Screening Method for Mycobactericidal Activity of Chemical Germicides That Uses Mycobacterium terrae Expressing a Green Fluorescent Protein Gene , 2001, Applied and Environmental Microbiology.

[16]  J. Logsdon,et al.  Genetic complementation in apicomplexan parasites , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[17]  D. Roos,et al.  Stable molecular transformation of Toxoplasma gondii: a selectable dihydrofolate reductase-thymidylate synthase marker based on drug-resistance mutations in malaria. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[18]  D. Roos,et al.  Inhibition of cytoplasmic and organellar protein synthesis in Toxoplasma gondii. Implications for the target of macrolide antibiotics. , 1995, The Journal of clinical investigation.

[19]  J. Boothroyd,et al.  Molecular analysis of the gene encoding the major surface antigen of Toxoplasma gondii. , 1988, Journal of immunology.

[20]  J. Klein,et al.  Infectious Diseases of the Fetus and Newborn Infant , 1983 .

[21]  T. Heier,et al.  Temperature-dependent Pharmacokinetics and Pharmacodynamics of Vecuronium , 2000, Anesthesiology.

[22]  D. Roos,et al.  Targeting of soluble proteins to the rhoptries and micronemes in Toxoplasma gondii. , 2001, Molecular and biochemical parasitology.

[23]  C. Schulz,et al.  TEMPERATURE‐DEPENDENT INOTROPIC EFFECTS OF LIGNOCAINE AND ETHANOL ON RAT HEART PAPILLARY MUSCLES , 1998, Clinical and experimental pharmacology & physiology.

[24]  J. Corbeil,et al.  A new reporter cell line to monitor HIV infection and drug susceptibility in vitro. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[25]  J. Boothroyd,et al.  Interconnection between organellar functions, development and drug resistance in the protozoan parasite, Toxoplasma gondii. , 1995, International journal for parasitology.

[26]  D. Roos,et al.  Molecular tools for genetic dissection of the protozoan parasite Toxoplasma gondii. , 1994, Methods in cell biology.

[27]  C. Chastang,et al.  Enzyme immunoassay to assess effect of antimicrobial agents on Toxoplasma gondii in tissue culture , 1988, Antimicrobial Agents and Chemotherapy.

[28]  J. Boothroyd,et al.  Gene replacement in Toxoplasma gondii with chloramphenicol acetyltransferase as selectable marker. , 1993, Science.

[29]  I. Coppens,et al.  Golgi biogenesis in Toxoplasma gondii , 2002, Nature.

[30]  Anton J. Hopfinger,et al.  Free energy force field (FEFF) 3D-QSAR analysis of a set of Plasmodium falciparum dihydrofolate reductase inhibitors , 2001, J. Comput. Aided Mol. Des..

[31]  J. Boothroyd,et al.  Adaptation of signature-tagged mutagenesis for Toxoplasma gondii: a negative screening strategy to isolate genes that are essential in restrictive growth conditions. , 2001, Molecular and biochemical parasitology.

[32]  B. Nare,et al.  Evaluation of a Cyclic GMP-Dependent Protein Kinase Inhibitor in Treatment of Murine Toxoplasmosis: Gamma Interferon Is Required for Efficacy , 2002, Antimicrobial Agents and Chemotherapy.

[33]  B. Luft,et al.  Toxoplasmic encephalitis in AIDS. , 1992, Clinical infectious diseases : an official publication of the Infectious Diseases Society of America.

[34]  J. Remington,et al.  Biology of Toxoplasma gondii. , 1993, AIDS.

[35]  D. Roos.  Primary structure of the dihydrofolate reductase-thymidylate synthase gene from Toxoplasma gondii. , 1993, The Journal of biological chemistry.

[36]  D. Roos,et al.  In vitro assays elucidate peculiar kinetics of clindamycin action against Toxoplasma gondii , 1995, Antimicrobial agents and chemotherapy.

[37]  C. Katlama,et al.  Treatment of central nervous system toxoplasmosis with pyrimethamine/sulfadiazine combination in 35 patients with the acquired immunodeficiency syndrome. Efficacy of long-term continuous therapy. , 1988, The American journal of medicine.

[38]  L. Sibley,et al.  Toxoplasma Invasion of Mammalian Cells Is Powered by the Actin Cytoskeleton of the Parasite , 1996, Cell.

[39]  J. Ferrandiz,et al.  Flow cytometric quantification of Toxoplasma gondii cellular infection and replication. , 1999, The Journal of parasitology.

[40]  J. Boothroyd,et al.  An rRNA mutation identifies the apicoplast as the target for clindamycin in Toxoplasma gondii , 2002, Molecular microbiology.

[41]  J. K. Frenkel,et al.  Acute toxoplasmosis. Effective treatment with pyrimethamine, sulfadiazine, leucovorin calcium, and yeast. , 1960, JAMA.

[42]  D. Roos,et al.  Insertional Tagging, Cloning, and Expression of the Toxoplasma gondii Hypoxanthine-Xanthine-Guanine Phosphoribosyltransferase Gene , 1996, The Journal of Biological Chemistry.

[43]  R. Heim,et al.  Understanding structure-function relationships in the Aequorea victoria green fluorescent protein. , 1999, Methods in cell biology.

[44]  D. Roos,et al.  Expression, selection, and organellar targeting of the green fluorescent protein in Toxoplasma gondii. , 1998, Molecular and biochemical parasitology.