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

描述（由申请人提供）：该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发病机制所必需的功能。 G.肠线虫属于已知最早的真核生物（双单胞菌）谱系，是人类和动物广泛分布的肠道寄生虫。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贾第药灭滴灵产生耐药性。这些观察结果强调了寻找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鞭毛虫生物学的不完全了解。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，并将测试选定的驱动蛋白（微管马达）如何促进有丝分裂、腹侧盘功能和组装以及成囊过程中的核配体和自混合。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），而新型驱动蛋白参与新的功能，例如成囊过程中的星体受精。我们开发了新的分子工具来研究贾第鞭毛虫的驱动蛋白功能。对 kin13 显性失活严格突变体的分析表明，它是间期和有丝分裂微管动力学的主要调节因子，并且与两个 kin2 代表一起调节鞭毛长度。我们的方法包括分析贾第鞭毛虫中的驱动蛋白定位，以确定进一步研究的候选者的优先顺序（目标 1），以及使用遗传和生化方法对选定的驱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。目标 2 中正在研究的贾第鞭毛虫有丝分裂驱动蛋白对于繁殖至关重要，并将成为激酶 ATP 酶活性小分子抑制剂化学遗传筛选的主要候选者。该分析还将为有丝分裂期间的驱动蛋白功能提供重要的进化视角。在具体目标 3 和 4 中，我们将研究导致细胞和核融合以及自混合的包囊过程中的核行为。 。自混合可能对于维持基因组稳定性和耐药性进化至关重要，因此是药物发现的目标。我们将确定驱动蛋白是否有助于细胞和核运动和融合（目标 3）以及融合后是否发生与同源重组相关的过程（目标 4）。这些结果将通过用含有整合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。有证据表明对目前的首选药物灭滴灵具有耐药性。这些观察结果强调了寻找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鞭毛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迄今未知的基本特征，包括细胞分裂和减数分裂，并为药物发现提供额外的目标。

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.