Adaptation of signature-tagged mutagenesis for Toxoplasma gondii: a negative screening strategy to isolate genes that are essential in restrictive growth conditions

The obligate intracellular parasite Toxoplasma gondii can infect virtually any nucleated cell in any warm-blooded host. Through the effort of many res earchers, we are beginning to learn what makes T. gondii such a successful protozoan parasite. A high throughput genetic screen that allows simultaneo us examination of a large panel of mutants would greatly facilitate a globa l investigation of this parasite. Signature-tagged mutagenesis uses a uniqu e DNA sequence to tag an individual mutant so that it can later be identifi ed within a pool. This system allows the efficient identification of parasi tes carrying mutations in genes that are essential for growth in restrictiv e but not permissive conditions. We have generated a bank of approximately 4900 signature-tagged T. gondii tachyzoites represented in 89 pools, each o f which contains 60 uniquely tagged mutant parasites. We have demonstrated the usefulness of this negative screening strategy with a tissue culture mo del for pyrimidine salvage using resistance to the pro-drug FUDR. Mutants t hat are defective for growth in any defined growth condition versus standar d tissue culture conditions can now be identified (eg, sensitive to a speci fic drug, growth in a specialized cell line, or growth within animals). (C) 2001 Elsevier Science B.V. All rights reserved.