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 缺陷甚至死亡。据估计，全球每年有超过 2 亿有症状病例的发病率。 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。 已有抗贾第药的报道。除了日益缺乏效率之外，一线和二线人员 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 负面影响。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。有鞭毛的滋养体增殖并定殖 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。虽然目前的治疗目标是 有鞭毛的滋养体，包囊可用于清除贾第鞭毛虫感染。成囊的调节 对包囊寄生虫的多样性知之甚少；因此，研究的重点是成囊 该途径具有基本的细胞和发育生物学意义，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 成囊计划。我们的初步研究表明，cAMP 升高是激活成囊所必需的。 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 营。使用 GlRac 进行的蛋白质-蛋白质相互作用研究发现了一个以前未表征的七- 属于 TOG（转运蛋白/视蛋白/GPCR）超家族的跨膜 PQ 环蛋白。击倒 该蛋白质导致成囊增加，表明它对成囊有负调节作用。我们把这个命名为 蛋白质 EcystR 在成囊过程中的作用。 EcystR 定位于活跃生长细胞的质膜， 但暴露于成囊刺激（高 pH 值和胆固醇消耗）会导致快速内化。包囊酶R 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。有关的 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， 可以调节发育。 EcystR 是揭示贾第鞭毛虫监管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 在引发成囊过程中的作用。这项工作将建立一个理解框架 从信号检测到成囊的成囊调节。最终，我们的目标是确定方法 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 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。