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

描述（由申请人提供）：本项目的目的是分析肠道寄生虫和基底真核生物贾第鞭毛虫的繁殖和发病机制所必需的功能。G.寄生虫是已知最早的真核生物分支，是人类和动物肠道内广泛存在的寄生虫。它是人类十大寄生虫之一，在美国，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血管药物Flagyl具有耐药性。这些观察结果强调了确定有限数量的已知抗贾第虫化合物的替代品的必要性，并强调了我们对贾第虫生物学的不完整知识。拟议的研究将建立基本原则，控制力产生的贾第虫微管细胞骨架，并将测试如何选择驱动蛋白（微管马达）促进有丝分裂，腹盘功能和组装，核融合和automixis在encystation。我们假设进化上保守的驱动蛋白参与保守的过程（例如，有丝分裂），而新的驱动蛋白参与新的功能，如包囊形成过程中的核配。我们已经开发了新的分子工具来研究驱动蛋白在贾第虫中的功能。kin 13的显性负刚性突变体的分析表明，它是一个主调节器的间期和有丝分裂微管动力学，并沿着与两个kin 2代表调节鞭毛长度。我们的方法包括在贾第鞭毛虫驱动蛋白本地化的分析，优先考虑候选人进行进一步研究（目标1），并选择驱动蛋白功能的深入研究，使用遗传和生化方法。(Aims 1-3）。我们将继续研究贾第虫有丝分裂的机制，将我们的分析扩展到活细胞。目的2中研究的心血管有丝分裂驱动蛋白对于生殖是必需的，并且将是用于激酶ATP酶活性的小分子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驱动蛋白的功能提供一个重要的进化视角。在具体目标3和4中，我们将研究导致细胞和核融合和自融合的成囊过程中的核行为。.自融合可能是维持基因组稳定性和耐药性进化所必需的，因此是药物发现的靶点。我们将确定驱动蛋白是否有助于细胞和核的运动和融合（目的3），以及融合后是否发生同源重组（目的4）。这些结果将通过用含有整合的选择标记的细胞感染动物模型来验证。公共卫生相关性：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。有证据表明对目前的首选药物Flagyl具有耐药性。这些观察结果强调了确定有限数量的已知抗贾第虫化合物的替代品的必要性，并强调了我们对贾第虫生物学的不完整知识。本文提出的研究将直接发现贾第虫生命周期的未知基本特征，包括细胞分裂和减数分裂，并为药物发现提供额外的靶点。

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.