Insulin-like signaling in parasitic nematode development

寄生线虫发育中的胰岛素样信号传导

• 批准号: 7149809
• 负责人: JAMES B LOK
• 金额: $ 39.25万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2011-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7149809
• 关键词: Nematoda; biological signal transduction; developmental genetics; green fluorescent proteins; host organism interaction; insulin receptor; intracellular parasitism; invertebrate embryology; microinjections; molecular cloning; polymerase chain reaction; regulatory gene

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.

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。在大多数这些感染中，第三幼虫阶段（L3 i）感染脊椎动物宿主。L3 i是过渡的、发育停滞的阶段，当暴露于终末宿主中存在的线索时，其被重新激活。寄生虫调节L3 i发育的机制仍不清楚，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，在自由生活的线虫秀丽隐杆线虫中调节L3形态发生的系统被很好地表征。这些系统包括胰岛素/IGF信号转导途径。拟议研究的总体目标是确定最近发现的类圆线虫中的胰岛素样途径是否也调节该寄生虫的发育，并通过类推，在寄生线虫中普遍存在。S.选择Stercoralis作为模型是因为这种蠕虫具有交替的自由生活周期，使人想起C.优雅的这个建议的具体目的是，首先，确定关键基因编码胰岛素样信号转导元件在粪类圆线虫。我们将完成我们的PI 3激酶编码基因pik-1，C的直系同源物的特性。elegans age-1，寻找S. daf-28是调节C.优美的其次，我们将研究S的胰岛素样信号途径中间产物的发育功能。粪虫这些研究的方法将强调异源基因转移到C.携带特定胰岛素途径突变的线虫。第三，我们将应用这些相同的方法来研究胰岛素途径中间体在调节S。粪虫最后，我们将描绘寄生虫特异性结构/功能的关系，在FKTF-1和描述细胞内运输的转录因子和daf-16直系同源物在发展过程中。将FKTF-1和FKTF-16的功能结构域组合的嵌合基因构建体将在突变体C中表达。elegans和S. stercoralis和对发育和亚细胞定位的影响进行评估。FKTF-1的亚细胞定位将在寄生虫生命周期的关键点确定，并与C. elegans的发展我们最近在S. stercoralis将允许我们使用C. elegans作为遗传替代物，并对S.粪菌本身