Signal perception and transduction regulating Giardia cyst formation

信号感知和转导调节贾第鞭毛虫包囊形成

• 批准号: 10604084
• 负责人: Alexander Richard Paredez
• 金额: $ 51.59万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-09-19 至 2027-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10604084
• 关键词: Amino Acid Transporter; Amino Acids; Awareness; Biology; Biosensor; Cell Cycle Arrest; Cell membrane; Cells; Cellular Assay; Cellular biology; Cessation of life; Cholesterol; Citrulline; Clinical Treatment; Complement; Complex; Cues; Cyclic AMP; Cyst; Data; Defect; Detection; Development; Developmental Biology; Diarrhea; Effectiveness; Energy-Generating Resources; Exposure to; Family; Foundations; Free Radicals; G-Protein-Coupled Receptors; Gastrointestinal Diseases; Generations; Genetic Transcription; Giardia; Giardia lamblia; Giardiasis; Goals; Growth; Guanosine Triphosphate Phosphohydrolases; Homologous Gene; Image; Incidence; Infection; Intervention; Intestines; Knowledge; Life Cycle Stages; Malabsorption Syndromes; Mastigophora; Membrane Proteins; Metabolic; Molecular; Molecular Conformation; Mutate; Names; Nutrient; Opsin; Oral; Parasites; Pathway interactions; Perception; Persons; Pharmaceutical Preparations; Phosphotransferases; Play; Process; Production; Proliferating; Proteins; Regulation; Reporting; Resistance; Risk; Role; Shapes; Signal Transduction; Starvation; Stimulus; Testing; Translating; Work; clinically relevant; design; detection of nutrient; gain of function; gastrointestinal; genetic analysis; gut colonization; improved; insight; interest; knock-down; nanoluciferase; novel; phosphoproteomics; prevent; programs; protein protein interaction; public health relevance; receptor; response; rho; rho GTP-Binding Proteins; sensor; side effect; spatiotemporal; therapeutic development; therapy resistant; trafficking; transmission process; treatment strategy; uptake

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.

项目总结/摘要 蓝氏贾第鞭毛虫，贾第虫病的病原体，导致胃肠道疾病，长期生长， 缺陷甚至死亡。据估计，全世界每年有2亿多有症状的病例。 值得关注的是，高达20%的贾第虫病病例对一线临床治疗具有耐药性，并且对所有主要的 抗心血管药物已有报道。除了越来越缺乏效率外，一线和二线- 线性硝基药物通过非特异性自由基的产生在细胞内起作用，因此具有高的 负面的副作用迫切需要更好地了解寄生虫的基本生物学， 最终设计改进的干预和治疗策略。鞭毛滋养体增殖定殖 肠道中的线索包括胆固醇饥饿和肠道末端的pH值增加 促进终末分化为包囊，包囊分离以进行传播。虽然目前的治疗针对的是 鞭毛虫滋养体，包囊可用于清除贾第虫感染。包囊形成的调控 在包囊寄生虫的多样性中知之甚少;因此，研究集中在包囊 这些信号通路具有基本的细胞和发育生物学意义，以及深刻的临床意义。 这个建议的目的是了解如何encystation线索被感知和转导启动 包囊化程序。我们的初步研究表明，升高的cAMP是激活包囊所必需的。 我们的初步研究确定了贾第虫唯一的Rho家族GTdR，GlRac，在调节 营与GlRac的蛋白质-蛋白质相互作用研究鉴定了先前未表征的七种- 跨膜PQ环蛋白属于TOG（转运蛋白/视蛋白/GPCR）超家族。敲低 该蛋白质导致包囊形成增加，表明其负调节包囊形成。我们把它命名为 蛋白EncystR在包囊形成中的作用。EncystR定位于活跃生长细胞的质膜， 但是暴露于包囊化刺激（高pH和胆固醇消耗）引起快速内化。EncystR 内化需要GlRac，支持这些蛋白质之间的功能关系。EncystR阴性 通过控制cAMP水平来调节成囊，但其机制仍有待确定。相关 PQ环蛋白是多功能氨基酸转运蛋白受体，已知其是pH响应性的， 可以规范发展。EncystR是一个令人兴奋的新切入点，可以揭示贾第虫的监管， 发展计划。 该项目的总体目标是描述EncystR和GlRac在调节cAMP和GlRac中的作用。 cAMP在引发成囊程序中的作用。这项工作将建立一个框架， 成囊从信号检测到成囊的调控。最终，我们的目标是确定 使正常的包囊形成程序短路，从而使细胞周期停滞和细胞骨架解体， 激活而不产生保护性的囊壁。这将清除感染而没有传播的风险。