INSULIN-LIKE SIGNALING IN PARASITIC NEMATODE DEVELOPMENT

描述（由申请人提供）：寄生线虫感染了世界上很大一部分人口，并造成了巨大的人类疾病。这些寄生虫以发育受阻的感染性幼虫（通常是第三阶段幼虫，L3i）的形式感染宿主，一旦进入宿主就会恢复发育。根据在秀丽隐杆线虫中广泛的相关发现，我们已经发现了明确的证据，表明胰岛素样（ILS）和类固醇核激素受体（NHR）信号在寄生线虫粪圆线虫感染前和感染过程中调节L3i的发育。在本次更新申请中，我们拟完成ILS对粪虫L3i发育调控的研究，并对保守的甾体NHR信号通路作为该寄生虫治疗靶点的潜力进行新的研究。我们的两个特定目的的第一个问题是胰岛素信号是否调节肠球菌L3i的形成和维持。具体来说，我们将确定胰岛素样受体激酶Ss-DAF-2和PI3激酶Ss-AGE-1是否阻断L3i的阻滞并促进其在宿主体内的发育再激活，以及胰岛素样肽Ss-ILP-6和Ss-ILP-7是否分别促进L3i的发育再激活和阻滞。我们的方法将包括分析基于这些分子在粪球菌中表达显性转基因构建体所产生的表型。特异性Aim 2将询问通过Ss-DAF-12 NHR的信号传导是否增强了S. stercoralis的调节ILS作用，以及这种小分子受体是否可以成为基于天然存在的类固醇或其类似物的化疗靶点。我们拟鉴定Ss-DAF-12的天然配体，并将其调节L3i阻滞和再激活的活性与秀丽线虫中的DAF-12配体D4-和d7 - dafachrononic acids （DA）进行比较。我们将使用化学抑制剂来探测内源性NHR DAF-12在粪球菌中的功能，并使用沙鼠感染模型来确定给药Ss-daf-12抑制剂或DA是否可以预防L3i的发展，清除成虫感染和/或阻断暴发性自身感染，这是人类圆线虫病的潜在致命并发症。这些实验将直接反映DAF-12 NHR作为寄生线虫化疗靶点的潜力。

DESCRIPTION (provided by applicant): Parasitic nematodes infect a significant proportion of the world's population and exact an enormous toll of human illness. These parasites infect their hosts as developmentally arrested infective larvae (usually third-stage larvae, (L3i) that resume development once they enter the host. Drawing upon extensive relevant findings in Caenorhabditis elegans, we have uncovered definitive evidence that insulin-like (ILS) and steroid-nuclear hormone receptor (NHR) signaling regulate L3i development before and during the infective process in the parasitic nematode Strongyloides stercoralis. In this renewal application, we propose to complete our studies on regulation of L3i development by ILS in S. stercoralis and initiate new research on the potential of a conserved steroid NHR signaling pathway as a therapeutic target in this parasite. The first of our two Specific Aims asks whether insulin signaling regulates formation and maintenance of L3i by S. stercoralis. Specifically we will determine whether the insulin-like receptor kinase Ss-DAF-2 and the PI3 kinase Ss-AGE-1 block arrest of L3i and promote their developmental reactivation in the host and whether insulin-like peptides Ss-ILP-6 and Ss-ILP-7 promote developmental reactivation and arrest of L3i, respectively. Our approach will involve analyzing phenotypes that result from expressing dominant transgene constructs based on these molecules in S. stercoralis. Specific Aim 2 will ask whether signaling through the Ss-DAF-12 NHR augments regulatory ILS effects in S. stercoralis and whether this small molecule receptor could be a target for chemotherapy based on naturally occurring steroids or their analogs. We propose to identify the natural ligands of Ss-DAF-12 and compare their activity in regulating L3i arrest and reactivation to the DAF-12 ligands from C. elegans, D4- and D7-dafachronic acids (DA). We will use chemical inhibitors to probe endogenous NHR DAF-12 function in S. stercoralis and use a gerbil model of infection to determine whether administered Ss-daf-12 inhibitors or DA can prevent development of L3i, clear adult worm infections and/or block fulminant autoinfection, a potentially fatal complication of human strongyloidiasis. These experiments will directly reflect the potential of DAF-12 NHR function as a chemotherapeutic target in parasitic nematodes.