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.
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. ? ? ?
