Signal perception and transduction regulating Giardia cyst formation

项目摘要/摘要 蓝氏贾第鞭毛虫是贾第鞭毛虫病的病原体，会导致胃肠功能紊乱，长期生长 缺陷甚至死亡。据估计，全球每年的有症状病例超过2亿例。 令人担忧的是，高达20%的贾第虫病病例对一线临床治疗具有抵抗力，对所有主要 已有抗心绞痛药物的报道。除了日益缺乏效力外，一线和二线- 线硝基药物通过非特异性自由基的产生在细胞内发挥作用，因此有很高的发病率 副作用很大。迫切需要更好地了解寄生虫的基本生物学，以便 最终设计改进的干预和治疗策略。有鞭毛的滋养体繁殖并定居 肠道，包括胆固醇饥饿和肠道末端PH值升高 促进末端分化为可分离传播的包囊。虽然目前的治疗针对的是 鞭毛滋养体、包囊作用可用于清除贾第鞭毛虫感染。包埋的管制 在包囊寄生虫的多样性中了解得很少；因此，研究集中在包囊 通路具有基本的细胞和发育生物学意义，也具有深远的临床意义。 这项建议的目的是了解如何感知和传递发声提示，以启动 加密程序。我们的初步研究表明，升高的cAMP是激活囊化所必需的。 我们的初步研究确定了贾第虫唯一的Rho家族GTP酶GlRac在调节 夏令营。与GlRac的蛋白质-蛋白质相互作用研究发现了一种以前未确定的七种- 跨膜PQ-loop蛋白，属于TOG(Transporter/opsin/GPCR)超家族。击倒 这种蛋白质导致囊化增加，表明它负向调节囊化。我们把这个命名为 EncystR蛋白在囊化过程中的作用。在生长活跃的细胞中，EncystR定位于质膜， 但暴露在囊化刺激(高pH值和胆固醇消耗)下，会导致快速内化。EncystR 内化需要GlRac，支持这些蛋白质之间的功能关系。负向EncystR 通过控制cAMP水平来调节包囊作用，但其机制尚不清楚。相关 PQ环蛋白是一种多功能氨基酸转运体受体，已知对pH有反应和 可以调节发展。EncystR是揭示贾第鞭毛虫的调节的一个令人兴奋的新切入点 发展规划。 这个项目的总体目标是描述EncystR和GlRac在调节cAMP和 CAMP在诱导结晶程序中的作用。这项工作将建立一个理解的框架 从信号检测到包埋的包埋调控。归根结底，我们的目标是确定 短路正常的囊化程序，使细胞周期停滞和细胞骨架解体 激活时不会产生保护性的囊壁。这将清除感染，而不会有传播的风险。

PROJECT SUMMARY/ABSTRACT Giardia lamblia, the causative agent of giardiasis, leads to gastrointestinal disorders, long-term growth defects and even death. Estimates place worldwide incidence at over 200 million symptomatic cases per year. Of concern, up to 20% of giardiasis cases are resistant to front-line clinical treatments, and resistance to all major antigiardial drugs has been reported. In addition to their increasing lack of effectiveness, front-line and second- line nitro drugs act intracellularly via non-specific free radical generation, and therefore have a high incidence of negative side effects. There is a critical need to better understand the basic biology of the parasite in order to ultimately design improved intervention and treatment strategies. Flagellated trophozoites proliferate to colonize the intestine where cues including cholesterol starvation and increased pH at the end of the intestinal tract promote terminal differentiation into cysts that detach for transmission. While current treatments target the flagellated trophozoites, encystation could be exploited to clear Giardia infections. The regulation of encystation is poorly understood across the diversity of encysting parasites; thus, studies focused on the encystation pathway are of fundamental cell and developmental biology interest, as well as profound clinical relevance. The aim of this proposal is to understand how encystation cues are perceived and transduced to initiate the encystation program. Our preliminary studies indicate that elevated cAMP is necessary to activate encystation. Our preliminary studies identified a role for Giardia’s sole Rho family GTPase, GlRac, in the regulation of cAMP. Protein-protein interaction studies with GlRac identified a previously uncharacterized seven- transmembrane PQ-loop protein that belongs to the TOG (transporter/opsin/GPCR) superfamily. Knockdown of this protein results in increased encystation indicating that it negatively regulates encystation. We named this protein EncystR for its role in encystation. EncystR localizes to the plasma membrane in actively growing cells, but exposure to encystation stimuli (high pH and cholesterol depletion), causes rapid internalization. EncystR internalization requires GlRac, supporting a functional relationship between these proteins. EncystR negatively regulates encystation through control of cAMP levels, but the mechanism remains to be determined. Related PQ loop proteins are multi-function amino acid transporter-receptors that are known to be pH responsive and can regulate development. EncystR is an exciting new entry point into uncovering the regulation of Giardia’s developmental program. The overall goal of this project is to delineate the roles of EncystR and GlRac in regulating cAMP and the role of cAMP in eliciting the encystation program. This work will establish a framework for understanding the regulation of encystation from signal detection to encystation. Ultimately, we aim to identify the means to short circuit the normal encystation program such that cell cycle arrest and cytoskeletal disassembly can be activated without producing a protective cyst wall. This would clear infections without the risk of transmission.