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

 描述(由调查人员提供)：据估计，致病原生生物溶组织内阿米巴每年造成50,000至100,000人死亡，是人类，特别是在发展中国家寄生虫病引起的发病率和死亡率的重要来源。溶解组织杆菌有一个简单的生活史，要么以感染性包囊形式存在，要么以 可移动的滋养体，可居住在人类结肠的厌氧范围内。虽然阿米巴病目前用甲硝唑治疗，但有必要开发更多有效抗组织溶血性埃希氏菌感染的药物。为了确定合适的靶点，重要的是首先对这种病原体的代谢和生理学有更深入的了解。溶组织线粒体依赖于一种不寻常的依赖于PPI的糖酵解途径作为产生ATP的主要手段。我们的长期目标是更好地了解溶组织埃希氏菌的代谢，因为这种独特的真核病原体在感染的不同阶段遇到不同的环境。该建议的目的是研究醋酸激酶(ACK)和ADP-形成乙酰辅酶A合成酶(ACD)的作用，这两种酶可能在PPI依赖的糖酵解途径中发挥关键作用。ACK主要是一种细菌酶，在完全可逆的反应中催化乙酰磷酸+ADP转化为醋酸酯+ATP。内阿米巴酶的独特之处在于，它催化乙酰磷酸+PI转化为乙酸酯+PPI，从而可能为糖酵解提供PPI。初步结果表明，ACK是必不可少的，与这一关键作用相一致。ACD催化乙酸乙酯和乙酰辅酶A的相互转化，只存在于少数真核微生物中，包括病原体疟原虫。如果用来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可能会在利用大肠微生物群产生的醋酸盐方面发挥作用。具体目标1是确定ACK和ACD中负责底物结合和催化的活性部位残基。根据结构分析和与密切相关的酶的类比，每个酶中的氨基酸残基将被定向改变。将分析这些酶变体，以确定这些变化对底物结合和催化的影响。具体目的2是确定ACK和ACD在溶组织埃希氏菌中的生理作用。反义RNA介导的基因沉默和一种新的RNAi拯救方法正在开发中，用于检测必需基因，以检测ACK和ACD在不同生长条件下的作用。将使用代谢图谱来确定以下物质的代谢影响 不同生长条件对野生型和RNAi/RNAi拯救突变体的影响。这些研究的结果将填补我们对内阿米巴新陈代谢知识的空白。当内阿米巴在感染期间穿过肠道时，这些酶在其拟议的作用中将在人类宿主的生长和生存中发挥重要作用，但这些作用需要得到证实。

 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.