The role of acetate fermentation in Entamoeba histolytica growth and infection

醋酸发酵在溶组织内阿米巴生长和感染中的作用

• 批准号: 9261575
• 负责人: CHERYL Jean INGRAM-SMITH
• 金额: $ 23.99万
• 依托单位: CLEMSON UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/9261575
• 关键词: Acetate-CoA Ligase; Acetates; Acetyl Coenzyme A; Actins; Active Sites; Anaerobic Bacteria; Bacteria; Basidiomycota; Biochemistry; Bioterrorism; Blastocystis; Categories; Cause of Death; Cells; Centers of Research Excellence; Cessation of life; Citric Acid Cycle; Coenzyme A; Colon; Cryptosporidium; Cyst; Developing Countries; Dose; Energy Metabolism; Energy Metabolism Pathway; Entamoeba; Entamoeba histolytica; Enzymatic Biochemistry; Enzymes; Evolution; Fermentation; Food Supply; Generations; Giardia; Glucose; Glycolysis; Goals; Growth; Heat-Shock Response; Human; Infection; Investigation; Life Cycle Stages; Metabolic; Metabolic Pathway; Microbe; Mitochondria; Morbidity - disease rate; National Institute of Allergy and Infectious Disease; Organism; Orthophosphate Dikinase Pyruvate; Oxidative Phosphorylation; Parasites; Parasitic Diseases; Pathogenicity; Pathway interactions; Phosphorylation; Phosphotransferases; Physiological; Physiology; Plasmodium; Play; Production; Property; Pyruvate; Pyruvate Kinase; Pyruvate synthase; RNA Interference; Reaction; Recombinants; Research; Research Personnel; Role; Source; Structure; Succinate-CoA Ligases; Water Supply; Work; acetyl phosphate; axenic culture; cell motility; combat; enzyme structure; fungus; in vivo; innovation; insight; interest; mortality; overexpression; pathogen; preference; pyruvate dehydrogenase; sugar; transcriptome

PROJECT SUMMARY The pathogenic protist Entamoeba histolytica is estimated to be responsible for 50,000 to 100,000 deaths each year and is a significant source of morbidity and mortality due to parasitic disease in humans, particularly in developing countries. This protist is also listed by NIAID as a category B priority pathogen with bioterrorism potential due to its low infectious dose and potential for dissemination through compromised food and water supplies. E. histolytica has a simple life cycle, existing in either an infectious cyst form or as motile trophozoites which can reside in the anaerobic confines of the human colon. This microbe is a scavenger that lacks ATP- producing mitochondria and relies on glycolysis as its primary pathway for generation of ATP. The PPi- dependent glycolytic pathway of Entamoeba has an increased net generation of ATP, which would be beneficial for organisms that lack a functional citric acid cycle and oxidative phosphorylation and must rely solely on substrate level phosphorylation to generate ATP from glucose breakdown. The proposed research examines the role of acetate fermentation in E. histolytica energy metabolism and physiology. Acetate is produced during growth in axenic culture, and the two enzymes of interest, acetate kinase (Ack) and ADP- forming acetyl-CoA synthetase (Acd), are both proposed to play a role in acetate production at the end of glycolysis. Acd is proposed to provide for generation of an additional ATP at the end of glycolysis. Recombinant E. histolytica Acd (EhAcd) has been shown to function well in both directions of the reaction, suggesting that this enzyme can operate in either direction in vivo, depending on the cell's metabolic needs. Recombinant E. histolytica Ack (EhAck) has been shown to have two unusual properties: production of PPi rather than ATP, and a strong preference for the acetate-forming direction of the reaction. Ack may thus provide a mechanism for supplying additional PPi for glycolysis under certain growth conditions that require a higher flux through this pathway. The two specific aims of this proposal are (1) to investigate the enzymology of EhAcd and EhAck using a structure-function approach; and (2) to determine the physiological roles of Acd and Ack in E. histolytica through transcriptome profiling and metabolic analysis of ACK and ACD RNAi and overexpression strains. The results from this research will provide a better understanding of a key metabolic pathway for ATP generation in this important eukaryotic pathogen.

项目概要 据估计，致病性原生生物溶组织内阿米巴会导致 50,000 至 100,000 人死亡 年，是人类寄生虫病发病率和死亡率的重要来源，特别是在 发展中国家。该原生生物还被NIAID列为具有生物恐怖主义的B类优先病原体 由于其低传染剂量和通过受损食物和水传播的潜力，因此具有潜力 补给品。溶组织内阿米巴有一个简单的生命周期，以传染性包囊形式或活动滋养体形式存在 它可以存在于人类结肠的厌氧范围内。这种微生物是一种缺乏 ATP 的清道夫 产生线粒体并依赖糖酵解作为其产生 ATP 的主要途径。 PPi- 内阿米巴依赖的糖酵解途径增加了 ATP 的净生成，这将是 对缺乏功能性柠檬酸循环和氧化磷酸化且必须依赖的生物体有益 仅通过底物水平磷酸化从葡萄糖分解产生 ATP。拟议的研究 检查醋酸发酵在溶组织内阿米巴能量代谢和生理学中的作用。醋酸盐是 在无菌培养物中生长过程中产生，以及两种感兴趣的酶，乙酸激酶 (Ack) 和 ADP- 形成乙酰辅酶A合成酶（Acd），两者都被认为在乙酸生产结束时发挥作用 糖酵解。 Acd 被提议在糖酵解结束时提供额外的 ATP 的产生。 重组溶组织内阿米巴 Acd (EhAcd) 已被证明在两个反应方向上都能发挥良好作用， 这表明这种酶可以在体内以任一方向运作，具体取决于细胞的代谢需求。 重组溶组织内阿米巴 Ack (EhAck) 已被证明具有两个不寻常的特性： 产生 PPi 而不是 ATP，并且强烈偏好反应的乙酸盐形成方向。因此Ack可以 提供了一种在某些生长条件下为糖酵解提供额外 PPi 的机制，这些条件需要 通过该途径的通量更高。该提案的两个具体目标是（1）研究 EhAcd 和 EhAck 使用结构函数方法； (2) 确定Acd和的生理作用 通过转录组分析和 ACK 和 ACD RNAi 的代谢分析，在溶组织内阿米巴中确认 Ack 过度表达菌株。这项研究的结果将有助于更好地理解关键代谢 这种重要的真核病原体中 ATP 生成的途径。