Role of insulin-like signaling in the hookworm infective process

描述（由申请人提供）：钩虫病仍然是全球最重要的传染病之一。近8亿人感染了一种或两种钩虫。严重钩虫感染造成的失血会导致缺铁性贫血，这在成长中的儿童、孕妇和老年人中尤其具有破坏性。在感染过程中，感染性幼虫（L3）遇到宿主特异性信号，重新激活其发育。在感染期间，这种自由生活的L3到寄生的L3的激活是一个非常重要的但知之甚少的事件。在相关的C.在elegans dauer中，胰岛素样信号传导（ILS）和转录因子β 16的磷酸化介导发育停滞的恢复。ILS是钩虫激活所必需的，并且已经在钩虫中鉴定出了一种IL-16分子。本项目将研究钩虫L3激活寄生的基本分子机制，特别是胰岛素信号和β-16在感染过程中的作用。目的1探讨钩虫L3激活过程中β-16的调控机制。将通过细胞分级分离和蛋白质印迹法测定非活化和活化L3中IL-16的磷酸化状态和细胞定位，并确定ILS抑制剂对这些参数的影响。目的2将确定ILS信号通过IL-16的激活相关钩虫基因的表达的影响。将使用定量PCR测量非活化、活化和ILS抑制的L3中已知发育调节基因的mRNA水平，以确定它们是否受IFN-16和ILS调节。目标3将确定胰岛素信号传导和β-16磷酸化是否是感染所必需的。将检测抑制的L3感染允许宿主的能力。胰岛素信号传导和β-16磷酸化被预测为成功感染所需。相关性：钩虫控制仅限于用驱虫药反复治疗，作为一种长期策略，这种方法会迅速再次感染，并可能产生抗药性。有效的重组疫苗至少还需要十年时间。开发新的控制方案，包括更好的抗原和新的药物靶点，取决于对钩虫感染的基本生物学的更详细的了解。该项目还将深入了解感染过程中的基本分子机制，并建立与人类重要的其他寄生线虫感染相关的寄生关系。

DESCRIPTION (provided by applicant): Hookworm disease continues to rank among the most important infectious diseases worldwide. Nearly 800 million people are infected with one or both species of hookworms. Blood loss in heavy hookworm infections causes iron-deficiency anemia that is especially devastating in growing children, pregnant women, and the elderly. During infection, the infective larva (L3) encounters a host-specific signal that re-activates its development. This activation of the free-living L3 to the parasitic L3 during infection is a critically important, but poorly understood, event. In the related C. elegans dauer, insulin-like signaling (ILS) and phosphorylation of the transcription factor DAF-16 mediates recovery from developmental arrest. ILS is required for hookworm activation, and a DAF-16 molecule has been identified in hookworms. This project will investigate the basic molecular mechanisms of hookworm L3 activation to parasitism, and specifically the role of insulin signaling and DAF-16, during infection. Aim 1 will investigate the mechanism of DAF-16 regulation during activation of hookworm L3. The phosphorylation status and cellular localization of DAF-16 in non-activated and activated L3 will be determined by cell fractionation and Western blots, and the effect of ILS inhibitors on these parameters established. Aim 2 will determine if ILS signaling through DAF-16 effects expression of activation-associated hookworm genes. Quantitative PCR will be used to measure mRNA levels of known developmentally regulated genes in non-activated, activated and ILS inhibited L3 to determine if they are regulated by DAF-16 and ILS. Aim 3 will determine if insulin signaling and DAF-16 phosphorylation are required for infection. Inhibited L3 will be tested for their ability to infect a permissive host. Insulin signaling and DAF-16 phosphorylation are predicted to be required for successful infection. Relevance: Hookworm control is limited to repeated treatment with anthelmintics drugs, which as a long-term strategy suffers from rapid re-infection and the potential for drug resistance. An effective recombinant vaccine is at least a decade away. The development of new control options, including better antigens and new drug targets, depends on a more detailed understanding of the basic biology of hookworm infection. This project will also provide insight into the basic molecular mechanisms operating during infection and the establishment of parasitic relationships that are relevant to other parasitic nematode infections of human significance.