GENETICS OF INVASION AND EGRESS IN TOXOPLASMA

弓形虫是一种严重危害世界各地人畜的病原体。除了它在发育中的胎儿中众所周知的致病机制外，近年来，这种原生动物寄生虫还通过它可能导致艾滋病患者的致命疾病而增加了它的恶名。目前，没有旨在影响人类健康的弓形虫疫苗，现有药物在长期治疗中有毒和/或耐受性差，这对艾滋病患者是必要的。因此，迫切需要对这种寄生虫的生物化学和细胞生物学进行进一步研究，以促进开发预防和治疗其引起的疾病的新方法。这项提议的目标是确定与入侵和出口有关的基因。我们的方法是设计出能够产生在这两个过程中有缺陷的突变体的选择策略。我们已经开发出一种生物素标记方法，可以将细胞内和细胞外的寄生虫相互分离。通过这种方式，在特定温度下未能入侵或在适当条件下未能外出的突变寄生虫可以被隔离。在侵袭研究中，我们选择分离温度敏感的突变体，因为这一过程对于弓形虫这样的专性细胞内病原体是必不可少的。在出口研究中，我们利用了其他人的观察结果，即当应用于受感染的培养物时，钙离子载体(如A23187)将诱导出口。我们已经成功地分离出对这种刺激没有反应的突变体，因此在离子载体处理后仍保持在细胞内。最后，我们还利用了这样一个事实，即对已经在细胞外的寄生虫进行电离团处理会使它们失去传染性，从而使我们能够选择对这种影响具有抵抗力的突变体。我们将扩大三种突变类型的数量，确定受影响的基因，并确定其蛋白产物的定位和可能发挥的作用。通过这种方式，我们将提供一些关于入侵和出口的关键过程，这些过程对于这种重要病原体的生长是必不可少的。

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.