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，致病性原生生物溶组织内阿米巴（Entamoeba histolytica）每年造成5万至10万人死亡。 是人类寄生虫病发病率和死亡率的重要来源，特别是在 发展中国家这种原生生物也被NIAID列为生物恐怖主义的B类优先病原体 由于其低感染剂量和通过受损食物和水传播的可能性， 用品. E.溶组织原虫有一个简单的生活史，以感染性包囊形式或活动的滋养体形式存在 其可以驻留在人结肠的厌氧范围内。这种微生物是缺乏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 有益于缺乏功能性柠檬酸循环和氧化磷酸化的生物体， 仅依赖于底物水平磷酸化以从葡萄糖分解产生ATP。拟议研究 研究了乙酸发酵在E.能量代谢和生理学。乙酸 在无菌培养物中生长期间产生的，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），都被认为在乙酸生产结束时发挥作用。 糖酵解Acd被提出用于在糖酵解结束时产生额外的ATP。 重组大肠已经显示溶组织菌Acd（EhAcd）在反应的两个方向上都很好地起作用， 这表明这种酶可以在体内以任何一个方向运作，这取决于细胞的代谢需要。 重组大肠溶组织菌Ack（EhAck）已显示具有两种不寻常的性质：产生PPi 而不是ATP，以及对反应的乙酸盐形成方向的强烈偏好。因此，Ack可以 提供了在某些生长条件下为糖酵解提供额外PPi的机制， 更高的通量通过这条途径。该建议的两个具体目标是：（1）研究 EhAcd和EhAck的结构-功能方法;和（2）确定Acd和EhAck的生理作用， 在E中确认。通过ACK和ACD RNAi的转录组谱分析和代谢分析， 过表达菌株。这项研究的结果将提供一个更好的理解一个关键的代谢 ATP生成途径在这一重要的真核病原体。