Insulin-like signaling in parasitic nematode development

项目总结/摘要 寄生线虫感染了世界上大约20%的人口， 人类疾病大多数寄生虫以发育停滞的感染性第三阶段感染宿主， 阶段幼虫（iL 3），一旦进入宿主就恢复发育。根据 线虫，我们已经表明，寄生线虫粪类圆线虫具有 显著改变胰岛素样（ILS）和类固醇核激素受体（NHR）信号传导， 在感染过程中调节IL 3的发育。在此续期申请中，我们建议 扩大我们的研究，iL 3的发展和形态发生的调节ILS和NHR信号， S. Stercoralis，部署我们的CRISPR/Cas9诱变新方法，以明确评估 基因功能和RNAseq，以确定由敲除关键基因引起的转录组学变化。 信号元素。具体目标1询问ILS是否调节L3 i的形成和发育， S. Stercoralis由ILS下游调节。为此，我们将淘汰 16，其编码ILS调节的转录因子;和Ss-daf-2，其编码胰岛素样 受体激酶在S.粪虫我们将对敲除蠕虫进行RNAseq，以确定全球 转录变化和涉及细胞色素P450基因的特异性变化， Ss-NHR-12配体的生物合成。目的2将分配功能的胰岛素样肽（ILP）， S. Stercoralis作为ILS的激动剂或拮抗剂，通过从设计为改变ILS的转基因中表达它们， 蠕虫转录的时间模式。目标2还将确定SS-12 NHR是否 信号反馈通过增强S. 粪虫目的3将采用体内和体外研究来验证Ss-NHR-12 NHR 信号调节S. stercoralis，延长它在自由生活的成年人和缩短它在 寄生的雌性所有这三个目标将有助于未来发展急需的新的 寄生线虫中靶向ILS和NHR信号传导的驱虫剂。阐明功能链接 这些途径之间的相互作用将使新的联合疗法，目标是两个途径， 添加或协同地阻断发育并加速蠕虫的死亡和排出。

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.