GENETICS OF INVASION AND EGRESS IN TOXOPLASMA

弓形虫是一种严重危害人类和家畜健康的病原体. 除了在发育中的胎儿中的众所周知的发病机制之外，近年来这种原生动物寄生虫通过它在艾滋病患者中引起的致命疾病而增加了它的恶名。目前，还没有设计用于影响人类健康的弓形虫疫苗，并且现有的药物在长期治疗中是有毒的和/或耐受性差的，这对于AIDS患者是必需的。 因此，迫切需要进一步研究这种寄生虫的生物化学和细胞生物学，以促进开发预防和治疗它引起的疾病的新方法。这项计划的目标是确定参与入侵和出口的基因。 我们的方法是设计选择策略，可以产生在这两个过程中有缺陷的突变体。 我们已经开发了一种生物素标记方法，可以将细胞内和细胞外寄生虫相互分选。 通过这种方式，可以分离出在特定温度下不能入侵或在适当条件下不能外出的突变寄生虫。 对于入侵研究，我们选择分离温度敏感的突变体，因为这一过程是必不可少的专性细胞内病原体，如弓形虫。 对于排出研究，我们利用了其他人的观察结果，即当应用于感染的培养物时，钙离子载体如A23187将诱导排出。 我们已经成功地分离出突变体，这些突变体对这种刺激没有反应，因此在离子载体处理后仍留在细胞内。最后，我们还利用了这样一个事实，即离子载体治疗已经在细胞外的寄生虫会使它们失去感染性，从而使我们能够选择对这种效应有抗性的突变体。 我们将扩大这三种突变类型的数量，确定受影响的基因，并确定其蛋白质产物的定位和可能发挥的作用。通过这种方式，我们将对这种重要病原体生长所必需的入侵和外出的关键过程提供一些见解。

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.