Entamoeba Metabolism: The Role of Acetate Kinase and ADP-Forming Acetyl-CoA Synthetase

内阿米巴代谢：乙酸激酶和 ADP 形成乙酰辅酶 A 合成酶的作用

基本信息

批准号：
9021770
负责人：
CHERYL Jean INGRAM-SMITH
金额：
$ 42.4万
依托单位：
CLEMSON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-04-01 至 2020-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9021770
关键词：
ATP Synthetase Complexes Acetate-CoA Ligase Acetates Acetyl Coenzyme A Active Sites Amebiasis Amino Acids Antisense RNA Attention Binding Biochemistry Blastocystis Catalysis Cause of Death Cells Cessation of life Citric Acid Cycle Colon Cryptosporidium Cyst Dependence Developing Countries Development Disease Entamoeba Entamoeba histolytica Environment Enzymes Essential Genes Ethanol Evolution Gene Silencing Generations Giardia Glucose Glycolysis Goals Growth Human Individual Infection Intestines Investigation Kinetics Knowledge Large Intestine Life Cycle Stages Mediating Metabolic Metabolic Pathway Metabolism Methanosarcina Methods Metronidazole Microbe Mitochondria Modification Morbidity - disease rate Output Oxidative Phosphorylation Parasites Parasitic Diseases Pathway interactions Phosphotransferases Physiological Physiology Plasmodium Play Production Property Pyrococcus Pyruvate RNA Interference Reaction Research Research Personnel Role Source Staging Structural Models Structure Succinate-CoA Ligases Variant Volatile Fatty Acids Work acetyl phosphate base combat enzyme structure insight interest metabolic profile microbiota mortality mutant novel pathogen public health relevance

项目摘要

DESCRIPTION (provided by investigator): 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. 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. Although amoebiasis is currently treated with metronidazole, development of additional agents effective against E. histolytica infection is warranted. In order to identify suitable targets, it is importat to first develop a more in- depth understanding of the metabolism and physiology of this pathogen. The amitochondriate E. histolytica relies on an unusual PPi-dependent glycolytic pathway as a primary means for ATP generation. Our long-term goal is to provide a better understanding of E. histolytica metabolism as this unique eukaryotic pathogen traverses the different environments encountered during various stages of infection. The objective of this proposal is to investigate the roles of acetate kinase (ACK) and ADP-forming acetyl-CoA synthetase (ACD), two enzymes that may play key roles in the PPi-dependent glycolytic pathway. ACK is primarily a bacterial enzyme that catalyzes the conversion of acetyl phosphate + ADP to acetate + ATP in a fully reversible reaction. The Entamoeba enzyme is unique in that it catalyzes the conversion of acetyl phosphate + Pi to acetate + PPi, and thus may function to supply PPi for glycolysis. Preliminary results have shown that ACK is essential, consistent with this critical role. ACD catalyzes the interconversion of acetate and acetyl-CoA and is present in only a handful of eukaryotic microbes including the pathogens Plasmodium. If used to break down acetyl-CoA produced from glycolysis, ACD's effect would be to increase ATP production. However, ACD may instead play a role in utilizing acetate produced by the microbiota in the large intestine. Specific Aim 1 is to identify active site residues in ACK and ACD responsible for substrate binding and catalysis. Amino acid residues in each enzyme will targeted for alteration based on structural analysis and analogy to closely related enzymes. The enzyme variants will be analyzed to determine the effects of these alterations on substrate binding and catalysis. Specific Aim 2 is to determine the physiological roles of ACK and ACD in E. histolytica. Antisense RNA-mediated gene silencing and a novel RNAi rescue method being developed for examination of essential genes will be used to examine the roles of ACK and ACD under different growth conditions. Metabolic profiling will be used to determine the metabolic effects of the different growth conditions on the wild type and RNAi/RNAi rescue mutants. The results of these studies will fill a gap in our knowledge of Entamoeba metabolism. These enzymes in their proposed roles would play an important role in growth and survival in the human host as Entamoeba traverses the intestinal tract during infection, but such roles need to be confirmed.

描述（由研究者提供）：据估计，致病性原生生物溶组织阿米巴每年导致 50,000 至 100,000 人死亡，并且是人类寄生虫病发病和死亡的重要来源，特别是在溶组织内阿米巴存在的发展中国家。简单的生命周期，以传染性包囊形式存在或作为可以驻留在人类结肠厌氧范围内的活动滋养体虽然阿米巴病目前使用甲硝唑，但有必要开发其他有效治疗溶组织阿米巴感染的药物。为了确定合适的靶标，重要的是首先开发更有效的药物。深入了解这种病原体的代谢和生理学，线粒体溶组织内阿米巴依赖于一种不寻常的 PPi 依赖性糖酵解途径。我们的长期目标是更好地了解溶组织内阿米巴的代谢，因为这种独特的真核病原体在感染的各个阶段会经历不同的环境。本提案的目的是研究乙酸盐的作用。 ACK 主要是一种细菌酶，在完全可逆反应中催化乙酰磷酸+ ADP 转化为乙酸+ ATP 内阿米巴酶的独特之处在于它催化乙酰磷酸+ Pi 转化为乙酸+ PPi，因此可以起到为糖酵解提供PPi 的作用。结果表明，ACK 是必不可少的，这与 ACD 催化乙酸盐和乙酸盐的相互转化相一致。乙酰辅酶A仅存在于少数真核微生物中，包括病原体疟原虫。如果用于分解糖酵解产生的乙酰辅酶A，ACD的作用将是增加ATP的产生，然而，ACD可能在利用乙酸中发挥作用。具体目标 1 是确定 ACK 和 ACD 中负责底物结合和催化的活性位点残基。根据结构分析和与密切相关的酶的类比，将针对每种酶中的氨基酸残基进行改变，以确定这些改变对底物结合和催化的影响。具体目标 2 是确定其生理作用。溶组织内阿米巴中的 ACK 和 ACD 介导的基因沉默和正在开发的用于检查必需基因的新型 RNAi 救援方法将用于检查 ACK 和 ACD 在不同生长条件下的作用。分析将用于确定代谢影响野生型和 RNAi/RNAi 拯救突变体的不同生长条件这些研究结果将填补我们对内阿米巴代谢的认识空白。这些酶的作用将在人类宿主的生长和生存中发挥重要作用。因为内阿米巴在感染过程中会穿过肠道，但这种作用需要得到证实。