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.

项目摘要 据估计，致病性的遗传性溶血症是造成50,000至100,000人死亡的造成的 一年，是人类寄生疾病引起的发病率和死亡的重要来源，特别是 发展中国家。 NIAID还将这种原生物列为B类优先病原体的生物恐怖主义 由于其低传染剂量和通过受损的食物和水的传播潜力而引起的潜力 补给品。 E. istolytica具有简单的生命周期，以感染性囊肿形式或运动滋养体存在 可以驻留在人类结肠的厌氧范围内。这个微生物是缺乏ATP-的清道夫 产生线粒体并依赖于糖酵解作为其产生ATP的主要途径。 PPI- Entamoeba的依赖性糖酵解途径的净产生增加了ATP，这将是 对缺乏功能性柠檬酸周期和氧化磷酸化的生物有益，必须依靠 仅基于底物水平磷酸化，从葡萄糖分解产生ATP。拟议的研究 研究醋酸盐发酵在E. istolytica Energy代谢和生理学中的作用。醋酸盐是 在轴原培养的生长过程中产生，乙酸激酶（ACK）和ADP-的两种酶 形成乙酰辅酶A合成酶（ACD）都建议在乙酸中发挥作用。 糖酵解。提议ACD在糖酵解结束时提供额外的ATP。 重组E. issolytica ACD（EHACD）已显示在反应的两个方向上都很好地发挥作用， 这表明该酶可以在体内沿任一个方向运行，具体取决于细胞的代谢需求。 重组E. histolytica ACK（EHACK）已证明具有两个不寻常的特性：PPI的产生 而不是ATP，并且强烈偏爱反应的乙酸盐形成方向。因此，ACK可能 在某些需要的生长条件下，提供了一种为糖酵解提供其他PPI的机制 通过这一路径较高的通量。该提案的两个具体目的是（1）研究的酶学 EHACD和EHACK使用结构功能方法； （2）确定ACD和 通过转录组分析和ACK和ACD RNAi和ACD RNAi的代谢分析以及ACD的ACK。 过表达菌株。这项研究的结果将更好地了解关键代谢 在这种重要的真核病原体中，ATP生成的途径。