背景:众所周知,蜕皮类固醇激素在节肢动物的发育和繁殖中起重要作用,由蜕皮类固醇受体介导。蜱是专性吸血节肢动物和病原体的载体。唾液腺在蜱的生长和繁殖以及病原体向脊椎动物宿主的传播中起着至关重要的作用。在滴答发展过程中,唾液腺经历由蜕皮类固醇激素触发的退化,并由细胞凋亡激活。然而,目前尚不清楚蜕皮类固醇受体和细胞凋亡如何调节唾液腺变性。这里,我们报告了功能性蜕皮类固醇受体(蜕皮激素受体[EcR]和超异酶[USP]的异二聚体),并探讨了蜕皮类固醇受体调节唾液腺变性的分子机制。
方法:从转录组获得RhEcR和RhUSP开放阅读框(ORF)的全长。RhEcR和RhUSP蛋白在细菌异源系统中表达,大肠杆菌。产生了针对合成肽的多克隆抗体,并且能够识别重组和天然蛋白质。采用实时定量PCR和Westernblot检测RhEcR的分布,RHUSP,和RhCaspases在嗜血杆菌器官中。蛋白质组学方法用于分析蜕皮类固醇受体的表达谱,RhCaspases,和其他蛋白质。为了分析蜕皮类固醇受体的功能,RNA干扰(RNAi)用于沉默成年雌性蜱中的基因。最后,通过在HEK293T细胞中的异源共表达试验鉴定了RhEcR和RhUSP的相互作用。
结果:我们从蜱嗜血杆菌唾液腺中鉴定出20-羟基蜕皮激素的功能性蜕皮激素受体(RhEcR/RhUSP)。RhEcR和RhUSP基因有三个和两个亚型,分别,并且属于核受体家族,但具有可变的N-末端A/B结构域。RhEcR基因沉默抑制血液摄食,阻止充血,和限制唾液腺变性,显示了RhEcR基因在蜱中的生物学作用。在蜕皮类固醇信号通路中,RhEcR沉默通过抑制caspase依赖性细胞凋亡来抑制唾液腺变性。哺乳动物HEK293T细胞中的异源表达表明RhEcR1与RhUSP1相互作用并诱导胱天蛋白酶依赖性细胞凋亡。
结论:这些数据表明,RhEcR在蜱生理中起着至关重要的作用,并代表了控制蜱和蜱传疾病的假定目标。
BACKGROUND: It is well established that ecdysteroid hormones play an important role in arthropod development and reproduction, mediated by ecdysteroid receptors. Ticks are obligate hematophagous arthropods and vectors of pathogens. The salivary gland plays an essential role in tick growth and reproduction and in the transmission of pathogens to vertebrate hosts. During tick development, the salivary gland undergoes degeneration triggered by ecdysteroid hormones and activated by apoptosis. However, it is unknown how the ecdysteroid receptor and apoptosis regulate salivary gland degeneration. Here, we report the functional ecdysteroid receptor (a heterodimer of the ecdysone receptor [EcR] and ultraspiracle [USP]) isolated from the salivary gland of the tick Rhipicephalus haemaphysaloides and explore the molecular mechanism of ecdysteroid receptor regulation of salivary gland degeneration.
METHODS: The full length of RhEcR and RhUSP open reading frames (ORFs) was obtained from the transcriptome. The RhEcR and RhUSP proteins were expressed in a bacterial heterologous system, Escherichia coli. Polyclonal antibodies were produced against synthetic peptides and were able to recognize recombinant and native proteins. Quantitative real-time PCR and western blot were used to detect the distribution of RhEcR, RhUSP, and RhCaspases in the R. haemaphysaloides organs. A proteomics approach was used to analyze the expression profiles of the ecdysteroid receptors, RhCaspases, and other proteins. To analyze the function of the ecdysteroid receptor, RNA interference (RNAi) was used to silence the genes in adult female ticks. Finally, the interaction of RhEcR and RhUSP was identified by heterologous co-expression assays in HEK293T cells.
RESULTS: We identified the functional ecdysone receptor (RhEcR/RhUSP) of 20-hydroxyecdysone from the salivary gland of the tick R. haemaphysaloides. The RhEcR and RhUSP genes have three and two isoforms, respectively, and belong to a nuclear receptor family but with variable N-terminal A/B domains. The RhEcR gene silencing inhibited blood-feeding, blocked engorgement, and restrained salivary gland degeneration, showing the biological role of the RhEcR gene in ticks. In the ecdysteroid signaling pathway, RhEcR silencing inhibited salivary gland degeneration by suppressing caspase-dependent apoptosis. The heterologous expression in mammalian HEK293T cells showed that RhEcR1 interacts with RhUSP1 and induces caspase-dependent apoptosis.
CONCLUSIONS: These data show that RhEcR has an essential role in tick physiology and represents a putative target for the control of ticks and tick-borne diseases.