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: 9453718
• 负责人: CHERYL Jean INGRAM-SMITH
• 金额: $ 23.17万
• 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/9453718
• 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.

项目总结 据估计，致病原生生物溶组织内阿米巴每个人造成50,000至10,000人死亡 是人类寄生虫病引起的发病率和死亡率的重要来源，尤其是在 发展中国家。该原生生物也被NIAID列为具有生物恐怖主义的B类优先病原体 由于其低感染量和通过受损害的食物和水传播的可能性而产生的潜在风险 补给。溶组织棘球绦虫的生活史很简单，要么以感染性包囊形式存在，要么以可移动的滋养体形式存在。 它可以存在于人类结肠的厌氧环境中。这种微生物是一种缺乏ATP的清道夫- 产生线粒体，依赖糖酵解作为其产生三磷酸腺苷的主要途径。PPI- 依赖内阿米巴的糖酵解途径增加了ATP的净生成，这将是 有益于缺乏功能性柠檬酸循环和氧化磷酸化的生物体，必须依赖于 仅在底物水平上进行磷酸化，以从葡萄糖分解中产生ATP。拟议的研究 研究了醋酸盐发酵在溶组织性肠杆菌能量代谢和生理中的作用。醋酸盐是 在无菌培养的生长过程中产生的，以及两种感兴趣的酶，醋酸激酶(Ack)和ADP-2。 形成乙酰-辅酶A合成酶(ACD)，都被认为在乙酸酯合成的末期起作用 糖酵解。ACD被认为是为了在糖酵解结束时产生额外的三磷酸腺苷。 重组溶组性肠杆菌ACD(EhAcd)已被证明在反应的两个方向上都发挥良好的作用， 这表明，这种酶在体内可以在两个方向上发挥作用，这取决于细胞的新陈代谢需求。 重组溶组性肠杆菌Ack(EhAck)具有两个不同寻常的性质：产生PPI 而不是三磷酸腺苷，并且强烈偏爱醋酸盐形成的反应方向。ACK可能因此 提供一种机制，在某些生长条件下为糖酵解提供额外的PPI，这些条件需要 更高的流量通过这一途径。这项建议的两个具体目标是：(1)研究 EhAcd和EhAck；以及(2)确定ACD和EhAck的生理作用 通过对ACK和ACD RNAi和ACD RNAi的转录组分析和代谢分析，确定了ACK在溶组织埃希菌中的表达 高表达菌株。这项研究的结果将提供对一种关键新陈代谢的更好的理解 在这种重要的真核病原体中产生ATP的途径。