Insulin-like signaling in parasitic nematode development

描述（由申请人提供）：寄生线虫使全世界数百万人患病或衰弱。在大多数感染中，第三幼虫期（L3i）感染脊椎动物宿主。L3i是过渡性的，发育受阻的阶段，当暴露于最终宿主中存在的线索时，它被重新激活。由于缺乏针对这些生物体的现代分子生物学方法，寄生虫调控L3i发育的机制尚不清楚。相比之下，调节秀丽隐杆线虫（Caenorhabditis elegans） L3形态发生的系统被很好地描述了。这些系统包括胰岛素/IGF信号转导通路。这项拟议研究的总体目标是确定最近在粪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，并以此类推，调节寄生线虫的发育。之所以选择粪虫作为模型，是因为这种蠕虫有一个交替的自由生活周期，让人想起秀丽隐杆线虫的持续发育。本研究的具体目的是：首先，确定粪圆线虫中编码胰岛素样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的表征，这是线虫age-1的同源基因，并寻找线虫中调节L3发育的胰岛素样配体daf-28的同源基因。其次，我们将研究肠球菌中胰岛素样信号通路中间体的发育功能。这些研究的方法将强调外源基因转移到携带特定胰岛素途径突变的秀丽隐杆线虫菌株中。第三，我们将运用这些相同的方法来研究胰岛素途径中间体在调节S. stercoralis寿命中的作用。最后，我们将描述FKTF-1的寄生虫特异性结构/功能关系，并描述该转录因子和daf-16同源物在发育过程中的细胞内运输。结合FKTF-1和DAF-16功能域的嵌合基因构建将在突变的秀丽隐杆线虫和粪线虫中表达，并评估其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时刻确定，并与线虫发育的类似点上的DAF-16进行比较。我们最近在粪虫转基因方面的成功将使我们能够利用秀丽隐杆线虫作为遗传替代物的实验与在粪虫本身中同源转基因构建的研究相结合。

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.