Kinesin function in mitosis and automixis in the widespread parasite Giardia inte

描述(申请人提供)：本项目的目标是分析肠道寄生虫和基础真核生物肠贾第鞭毛虫的繁殖和致病所必需的功能。肠圆线虫属于已知的最早的真核生物分支，是一种广泛存在于人和动物体内的肠道寄生虫。它是人类十大寄生虫之一，在美国，据估计每年发生数百万例。此外，最近有体外证据表明，对广泛使用的抗心绞痛药物Flagyl具有耐药性。这些观察结果强调了寻找已知抗贾第鞭毛虫化合物数量有限的替代品的必要性，并强调了我们对贾第虫生物学的不完全了解。这项拟议的研究将建立控制贾第鞭毛虫微管细胞骨架中力产生的基本原理，并将测试选定的运动蛋白(微管发动机)如何促进有丝分裂、腹盘功能和组装，以及囊化过程中的核受精和自身化。我们假设进化上保守的Kinesin参与保守的过程(例如，有丝分裂)，而新的Kinesin参与新的功能，如囊化过程中的核配子。我们已经开发了新的分子工具来研究贾第虫的动蛋白功能。对一个显性负刚性突变体kin13的分析表明，它是间期和有丝分裂微管动力学的主要调节者，并与两个kin2代表一起调节鞭毛长度。我们的方法包括分析运动蛋白在贾第虫中的定位，以确定进一步研究的优先候选对象(目标1)，以及使用遗传和生化方法对选定的运动蛋白功能进行深入研究。(目标1-3)。我们将继续研究贾第鞭毛虫有丝分裂的机制，将我们的分析扩展到活细胞。在AIM 2中正在研究的有丝分裂激动素对生殖是必不可少的，并将成为小分子激酶ATPase活性抑制剂的化学遗传筛选的主要候选者。这一分析也将为有丝分裂过程中动蛋白的功能提供一个重要的进化视角。在特定的目标3和4中，我们将研究导致细胞和核融合和自体融合的囊化过程中的核行为。。自交会可能是维持基因组稳定性和耐药性进化的关键，因此也是药物发现的目标。我们将确定动蛋白是否有助于细胞和核的移动和融合(目标3)，以及与同源重组相关的过程是否在融合后发生(目标4)。这些结果将通过用含有整合的可选择标记的细胞感染动物模型来验证。公共卫生相关性：肠贾第鞭毛虫是人类十大寄生虫之一，在美国，估计每年发生数百万例。有证据表明，目前的首选药物弗拉吉具有抗药性。这些观察结果强调了寻找已知抗贾第鞭毛虫化合物数量有限的替代品的必要性，并强调了我们对贾第虫生物学的不完全了解。这里提出的研究将直接发现贾第鞭毛虫生命周期的未知基本特征，包括细胞分裂和减数分裂，并为药物发现提供更多的靶点。

DESCRIPTION (provided by applicant): The goal of this project is to analyze functions essential to the reproduction and pathogenesis of the intestinal parasite and basal eukaryote, Giardia intestinalis. G. intestinalis belongs to the earliest known diverging lineage of eukaryotes (diplomonads), and it is a widespread intestinal parasite of humans and animals. It is one of the ten major parasites in humans, and in the USA, it is estimated that several million cases occur annually. Further, there is recent in vitro evidence of drug resistance to the widely used antigiardial, Flagyl. These observations underscore the necessity of identifying alternatives to the limited number of known antigiardial compounds, and highlight our incomplete knowledge of Giardia biology. The proposed research will establish fundamental principles governing force generation in the Giardia microtubule cytoskeleton, and will test how selected kinesins (microtubule motors) promote mitosis, ventral disc function and assembly, and karyogamy and automixis during encystation. We hypothesize that the evolutionarily conserved kinesins are involved in conserved processes (for example, mitosis), whereas the novel kinesins are involved in novel functions such askaryogamy during encystation. We have developed new molecular tools for studying kinesin function in Giardia. Analysis of a dominant negative rigor mutant of kin13 demonstrates that it is a master regulator of interphase and mitotic microtubule dynamics, and along with the two kin2 representatives regulates flagellar length. Our approach includes an analysis of kinesin localization in Giardia to prioritize candidates for further study (Aim 1), and an in-depth study of selected kinesin functions using a genetic and biochemical approach. (Aims 1-3). We will continue to investigate the mechanism of mitosis in Giardia, extending our analysis to living cells. The giardial mitotic kinesins under investigation in aim 2 are essential for reproduction and will be prime candidates for chemical genetic screens for small molecule inhibitors of kinase ATPase activity. This analysis will also provide an important evolutionary perspective on kinesin function during mitosis. In specific aims 3 and 4 we will investigate nuclear behavior during encystation that leads to cell and nuclear fusion and automixis. . Automixis may be essential for maintenance of genome stability and evolution of drug resistance, and therefore is a target for drug discovery. We will determine whether kinesins contribute to cell and nuclear movement and fusion (aim 3) and whether processes related to homologous recombination occur after fusion (aim 4). These results will be validated by infecting animal models with cells containing integrated selectable markers. PUBLIC HEALTH RELEVANCE: Giardia intestinalis is one of the ten major parasites in humans, and in the USA, it is estimated that several million cases occur annually. There is evidence of drug resistance to Flagyl, the current drug of choice. These observations underscore the necessity of identifying alternatives to the limited number of known antigiardial compounds, and highlight our incomplete knowledge of Giardia biology. The research proposed here will directly discover as yet unknown essential features of the Giardia life cycle, including cell division and meiosis and offer additional targets for drug discovery.