Insulin-like signaling in parasitic nematode development

寄生线虫使全世界数以百万计的人患病或虚弱。在这些感染中的大多数， 第三幼虫阶段(L3I)感染脊椎动物宿主。L3I是过渡性的、发展受阻的阶段， 当暴露于最终宿主中存在的提示时，它们被重新激活。寄生虫的致病机制 调节L3I的发育仍不清楚，这主要是由于缺乏现代分子生物学方法来 这些有机体。相比之下，调节自由生活线虫L3形态发生的系统 秀丽隐杆线虫的特征很好。这些系统包括胰岛素/胰岛素样生长因子信号转导。 路径。这项拟议研究的总体目标是确定最近发现的一种胰岛素样物质 类圆线虫中的途径也调节该寄生虫的发育，以此类推，在寄生虫中也是如此 一般都是线虫。之所以被选为模型，是因为这种虫有另一种自由生活的方式 周期，让人想起线虫的持续发展。这项建议的具体目的是，第一， 寻找编码类胰岛素信号转导元件的关键基因。我们会 完成我们对PI3激酶编码基因pik-1的表征，pik-1是线虫AGE-1的同源基因，以及 寻找调控线虫L3发育的胰岛素样配体daf-28的S。 其次，我们将研究S。胰岛素样信号通路中间产物的发育功能。 菊科植物。这些研究的方法将强调将异源基因转移到线虫品系中 携带特定的胰岛素途径突变。第三，我们将使用这些相同的方法来调查角色 胰岛素途径中间产物在调节粪肠蠕虫寿命中的作用。最后，我们将描述寄生虫- FKTF-1的特殊结构/功能关系，并描述该转录在细胞内的运输 发育过程中的因子和daf-16同源基因。嵌合基因构建，结合功能域 FKTF-1和DAF-16在突变线虫和杂色线虫中的表达及其对发育的影响 和亚细胞定位评估。FKTF-1的亚细胞定位将在#年的关键点确定 并与DAF-16在线虫发育过程中的类似点进行了比较。我们的 最近在司氏链霉菌转基因方面的成功将使我们能够扩大使用线虫作为 一种基因替代物，研究了紫珊瑚本身的同源转基因结构。

Parasitic nematodes sicken or debilitate millions of personsworldwide. In the majority of these infections, the third larval stage (L3i) infects the vertebrate host. L3i are transitional, developmental^ arrested stages, which are reactivated when exposed to cues present in the definitive host. Mechanisms by which parasites regulate L3i development remain unclear, due largely to the lack of modern molecular biological methods for these organisms. By contrast, systems regulating L3 morphogenesis in the free-living nematode Caenorhabditis elegans are well characterized. These systems include an insulin/IGF signal transduction pathway. The overall goal of the proposed study is to ascertain whether a recently discovered insulin-like pathway in Strongyloides stercoralis also regulates development in that parasite and, by analogy, in parasitic nematodes generally. S. stercoralis was chosen as a model because this worm has an alternate free-living cycle, reminiscent of continuous development in C. elegans. The specific aims of this proposal are, first, to identify key genes encoding insulin-like signal transduction elements in Strongyloides stercoralis. We will complete our characterization of the PI3 kinase-encoding gene pik-1, an ortholog of C. elegans age-1, and seek the S. stercoralis ortholog of daf-28, the insulin-like ligand regulating L3 development in C. elegans. Second, we will investigate the developmental function of insulin-like signal pathway intermediates from S. stercoralis. Methods for these studies will stress heterologous gene transfer into strains of C. elegans carrying specific insulin pathway mutations. Third, we will apply these same methods to investigate the roles of insulin pathway intermediates in regulating lifespan in S. stercoralis. Finally, we will delineate parasite- specific structure/function relationships in FKTF-1 and describe intracellular trafficking of this transcription factor and daf-16 ortholog during development. Chimeric gene constructs, combining functional domains of FKTF-1 and DAF-16 will be expressed in mutant C. elegans and in S. stercoralis and effects on development and subcellular localization assessed. Subcellular localization of FKTF-1 will be determined at key points in the parasite life cycle and compared to that of DAF-16 at analogous points in C. elegans' development. Our recent success with transgenesis in S. stercoralis will allow us to augment experiments using C. elegans as a genetic surrogate with studies of homologous transgene constructs in S. stercoralis itself.