Insulin-like signaling in parasitic nematode development

项目摘要/摘要 寄生线虫感染了大约20%的世界人口，并造成了巨大的损失 人类疾病。这些寄生虫中的大多数感染它们的宿主时，都是发育迟缓的感染性三分之一- 幼虫(IL3)进入寄主后恢复发育的阶段。参考了在 线虫Canowhabditis elegans，我们已经证明了寄生的斯特氏线虫具有 显著改变胰岛素样(ILS)和类固醇核激素受体(NHR)信号转导 在感染过程中调节iL3的发育。在这份续期申请中，我们建议 扩大我们对ILS和NHR信号对iL3发育和形态发生调控的研究 利用我们的新方法对CRISPR/Cas9突变进行明确评估 基因功能和RNAseq用于确定Key基因敲除引起的转录变化 信令元素。具体目标1问ILS是否调节L3I的形成和发展，以及 骨质炎链球菌下游受ILS调控的基因有哪些。为此，我们将摧毁党卫军-- 16，编码ILS调节的转录因子和SS-daf-2，编码类胰岛素 斯特氏链霉菌中的受体激酶。我们将对敲除蠕虫进行RNAseq测试，以确定全球 转录变化和涉及细胞色素P450基因的特异转录变化 SS-DAF-12 NHR配体的生物合成AIM 2将为胰岛素样肽(ILPS)分配功能 作为ILS的激动剂或拮抗剂，通过表达设计用于改变 蠕虫体内ILP转录的时间模式。AIM 2还将确定SS-DAF-12 NHR 信号反馈通过增强刺激性ILPS的表达对ILS进行正向调节。 菊科植物。Aim 3将采用体内和体外研究来检验SS-DAF-12 NHR的假设 信号转导调节了司体珊瑚的寿命，延长了自由生活的成虫的寿命，缩短了寿命 寄生的雌性。所有这三个目标都将有助于未来发展急需的新的 针对寄生线虫ILS和NHR信号的驱虫剂。阐明功能链接 在这些途径之间将实现新的联合疗法，针对这两个途径，作用 相加或协同作用，以阻止发育，加速成虫的死亡和排出。

PROJECT SUMMARY/ABSTRACT Parasitic nematodes infect roughly 20% of the world's population and exact an enormous toll in human illness. Most of these parasites infect their hosts as developmentally arrested infective third- stage larvae (iL3) that resume development once they enter the host. Drawing upon findings in Caenorhabditis elegans, we have shown that the parasitic nematode Strongyloides stercoralis has significantly modified insulin-like (ILS) and steroid-nuclear hormone receptor (NHR) signaling to regulate iL3 development during the infective process. In this renewal application, we propose to expand our studies of regulation of iL3 development and morphogenesis by ILS and NHR signaling in S. stercoralis, deploying our new method for CRISPR/Cas9 mutagenesis to unambiguously assess gene function and RNAseq to determine transcriptomic changes resulting from knockout of key signaling elements. Specific Aim 1 asks whether ILS regulates formation and development of L3i, and what genes in S. stercoralis are regulated by ILS downstream. To this end, we will knock out Ss-daf- 16, which encodes an ILS-regulated transcription factor and Ss-daf-2, which encodes the insulin-like receptor kinase in S. stercoralis. We will subject knockout worms to RNAseq to ascertain global transcriptional changes and specific ones involving cytochrome P450 genes that could act in biosynthesis of Ss-DAF-12 NHR ligands. Aim 2 will assign functions to insulin-like peptides (ILPs) in S. stercoralis as agonists or antagonists of ILS by expressing them from transgenes designed to alter temporal patterns of ilp transcription in the worms. Aim 2 will also determine whether Ss-DAF-12 NHR signaling feeds back to positively regulate ILS by enhancing expression of agonistic ILPs in S. stercoralis. Aim 3 will employ vivo and in vitro studies to test the hypothesis that Ss-DAF-12 NHR signaling regulates lifespan in S. stercoralis, lengthening it in free-living adults and shortening it in parasitic females. All three aims will contribute to future development of much needed new anthelmintics targeting ILS and NHR signaling in parasitic nematodes. Elucidating functional links between these pathways will enable new combination therapies that target both pathways, acting additively or synergistically to block development and accelerate death and expulsion of adult worms.