Toxoplasma gondii is a serious pathogen of humans and livestock world- wide. In addition to its well-known pathogenesis in the developing fetus, in recent years this protozoan parasite has increased its notoriety through the fatal disease it can cause in AIDS patients. Currently, there is no vaccine for Toxoplasma that is designed to impact human health and the existing drugs are toxic and/or poorly tolerated in long term treatment, as is necessary with AIDS patients. There is, thus, an urgent need for further research in the biochemistry and cell biology of this parasite to facilitate development of new means for preventing and treating the disease it causes. The goal of this proposal is to identify the genes involved in invasion and egress. Our approach is to devise selection strategies that can yield mutants defective in these two processes. We have developed a biotin-tagging method that allows intracellular and extracellular parasites to be sorted from one another. In this way, mutant parasites that fail to invade at a particular temperature or fail to egress under appropriate conditions can be isolated. For the invasion studies, we have chosen to isolate temperature-sensitive mutants since this process is essential for an obligate-intracellular pathogen like Toxoplasma. For the egress studies, we have exploited the observation of others that calcium ionophores such as A23187 will induce egress when applied to infected cultures. We have successfully isolated mutants that fail to respond to this stimulus and thus remain intracellular upon ionophore-treatment. Finally, we have also exploited the fact that ionphore-treatment of parasites that are already extracellular causes them to lose infectivity thus enabling us to select for mutants resistant to this effect. We will expand the numbers for each of the three mutant types, identify the affected genes and determine the localization and possible role played by their protein products. In this way, we will provide some insight into the critical processes of invasion and egress that are essential for growth of this important pathogen.
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