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: 10707172
• 负责人: Alexander Richard Paredez
• 金额: $ 50.74万
• 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/10707172
• 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; Cytoskeleton; Data; Defect; Detection; Development; Developmental Biology; Diarrhea; Effectiveness; Energy-Generating Resources; Environment; 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; Membrane Proteins; Metabolic; Molecular; Molecular Conformation; Mutate; Names; Nutrient; Opsin; Oral; Parasites; Pathway interactions; Perception; Persons; Pharmaceutical Preparations; Phosphotransferases; Process; Production; Proliferating; Proteins; Regulation; Reporting; Resistance; Risk; Role; Shapes; Signal Transduction; Starvation; Stimulus; Testing; Translating; Work; clinically relevant; design; detection of nutrient; drug action; gain of function; gastrointestinal; genetic analysis; gut colonization; improved; insight; interest; knock-down; loss of function; nanoluciferase; novel; overexpression; phosphoproteomics; prevent; programs; protein protein interaction; public health relevance; receptor; response; rho; rho GTP-Binding Proteins; sensor; side effect; spatiotemporal; therapeutic development; 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.

项目概要/摘要 贾第鞭毛虫病的病原体，会导致胃肠道疾病、长期生长 据估计，全球每年有超过 2 亿例有症状病例。 值得关注的是，高达 20% 的贾第鞭毛虫病病例对一线临床治疗具有耐药性，并且对所有主要的治疗方法均具有耐药性。 据报道，抗贾第药除了越来越缺乏疗效外，一线和二线药物也越来越缺乏疗效。 硝基药物通过非特异性自由基产生在细胞内发挥作用，因此发生率很高 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。 肠道的线索包括胆固醇饥饿和肠道末端 pH 值升高 促进终末分化为分离传播的囊肿。 有鞭毛的滋养体，可利用包囊来清除贾第鞭毛虫感染。 人们对包囊寄生虫的多样性知之甚少，因此，研究重点是包囊寄生虫； 通路具有基础细胞和发育生物学的意义，以及深刻的临床相关性。 该提案的目的是了解如何感知和转换成囊线索以启动 我们的初步研究表明，升高的 cAMP 是激活成囊细胞所必需的。 我们的初步研究确定了贾第鞭毛虫唯一的 Rho 家族 GTPase GlRac 在调节中的作用 cAMP。与 GlRac 的蛋白质-蛋白质相互作用研究发现了以前未表征的七- 属于 TOG（转运蛋白/视蛋白/GPCR）敲低超家族的跨膜 PQ 环蛋白。 这种蛋白质导致成囊增加，表明它对成囊有负调节作用，我们将其命名为。 蛋白质 EcystR 在成囊中的作用位于活跃生长的细胞的质膜上， 但暴露于成囊刺激（高 pH 值和胆固醇消耗）会导致快速内化。 内化需要 GlRac，支持这些蛋白质之间的功能关系。 通过控制 cAMP 水平来调节成囊，但其机制仍有待确定。 PQ 环蛋白是多功能氨基酸转运蛋白受体，已知具有 pH 响应性， EcystR 是揭示贾第鞭毛虫调控的一个令人兴奋的新切入点。 发展计划。 该项目的总体目标是描述 EcystR 和 GlRac 在调节 cAMP 和 cAMP 在引发成囊计划中的作用这项工作将建立一个理解框架。 最终，我们的目标是确定从信号检测到成囊的方法。 缩短正常的成囊程序，使细胞周期停滞和细胞骨架分解可以被 激活而不产生保护性囊肿壁，这将清除感染而没有传播风险。