Insulin-like signaling in parasitic nematode development

描述(申请人提供)：寄生线虫使全世界数百万人患病或虚弱。在大多数这些感染中，第三幼虫阶段(L3I)感染脊椎动物宿主。L3I是过渡性的、发育停滞的阶段，当接触到最终宿主中存在的线索时，L3I会被重新激活。寄生虫调节L3I发育的机制尚不清楚，这主要是由于缺乏针对这些生物体的现代分子生物学方法。相比之下，调节自由生活线虫L3形态发生的系统有很好的特征。这些系统包括胰岛素/胰岛素样生长因子信号转导通路。这项拟议研究的总体目标是确定最近在类圆线虫中发现的胰岛素样途径是否也调节该寄生虫的发育，以此类推，在一般寄生线虫中也是如此。之所以被选为模型，是因为这种蠕虫有一个交替的自由生活周期，这让人想起线虫的持续发展。这项建议的具体目的是，首先，确定编码类胰岛素信号转导元件的关键基因。我们将完成对PI3激酶编码基因pik-1的鉴定，该基因是线虫AGE-1的同源基因，并寻找调控线虫L3发育的胰岛素样配体daf-28的同源基因。其次，我们将研究类胰岛素信号通路中间产物的发育功能。这些研究的方法将强调将异源基因转移到携带特定胰岛素途径突变的线虫品系中。第三，我们将应用这些相同的方法来研究胰岛素途径中间产物在调节斯特氏链球菌寿命中的作用。最后，我们将描述FKTF-1中寄生虫特有的结构/功能关系，并描述该转录因子和daf-16同源基因在发育过程中的细胞内运输。结合FKTF-1和DAF-16功能区的嵌合基因构建将在突变的线虫和杂色线虫中表达，并评估对发育和亚细胞定位的影响。FKTF-1的亚细胞定位将在寄生虫生命周期的关键点确定，并在线虫发育的类似时间点与DAF-16进行比较。我们最近在司体珊瑚转基因方面的成功将使我们能够利用线虫作为基因替代品，通过研究司体珊瑚本身的同源转基因结构来扩大实验。

DESCRIPTION (provided by applicant): Parasitic nematodes sicken or debilitate millions of persons worldwide. In the majority of these infections, the third larval stage (L3i) infects the vertebrate host. L3i are transitional, developmentally 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.