Complementation of an Escherichia coli adhE mutant by the Entamoeba histolytica EhADH2 gene provides a method for the identification of new antiamebic drugs.
Complementation of an Escherichia coli adhE mutant by the Entamoeba histolytica EhADH2 gene provides a method for the identification of new antiamebic drugs.
复制标题
溶组织内阿米巴 EhADH2 基因与大肠杆菌 adhE 突变体的互补提供了一种鉴定新抗阿米巴药物的方法。
DOI：
10.1073/pnas.93.13.6464
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发表时间：
1996
影响因子：
11.1
通讯作者：
StanleyJr,SL
中科院分区：
文献类型：
--
作者：
Yong,TS;Li,E;Clark,D;StanleyJr,SL
致病性原生动物寄生虫溶组织内阿米巴（Entamoeba histolytica）是阿米巴痢疾和阿米巴肝脓肿的病因，是一种专性厌氧菌，从葡萄糖发酵成乙醇中获得能量，丙酮酸和乙酰辅酶A作为中间产物。我们已经分离到EhADH 2，这是一个关键酶在这一途径中，这是一个NAD+和Fe 2+依赖的双功能酶，乙醛脱氢酶和乙醇脱氢酶的活动。EhADH 2是新定义的原核多功能酶家族中唯一已知的真核成员，该家族包括大肠杆菌AdhE酶，一种大肠杆菌厌氧生长所需的酶。杆菌由于EhADH 2在阿米巴发酵途径中的关键作用以及缺乏已知的EhADH 2酶的真核同源物，EhADH 2代表了抗阿米巴化疗的潜在靶点。然而，筛选抗阿米巴活性的化合物受到溶组织内菌大规模生长的成本和体外定量药物功效的困难的阻碍。为了解决这个问题，我们在大肠杆菌突变株中表达了EhADH 2基因。大肠杆菌携带adhE基因的缺失。EhADH 2的表达恢复了突变体E.大肠杆菌菌株在厌氧条件下生长。通过筛选具有抑制大肠杆菌厌氧生长能力的化合物，coli/EhADH 2菌株，建立了一种快速鉴定具有抗EhADH 2活性化合物的方法。在抗寄生虫药物的初步筛选过程中，使用细菌绕过寄生虫培养的需要，可以大大简化和降低识别有效对抗寄生虫病的新治疗药物的成本。
The pathogenic protozoan parasite Entamoeba histolytica, the cause of amebic dysentery and amebic liver abscess, is an obligate anaerobe, and derives energy from the fermentation of glucose to ethanol with pyruvate and acetyl coenzyme A as intermediates. We have isolated EhADH2, a key enzyme in this pathway, that is a NAD+- and Fe2+-dependent bifunctional enzyme with acetaldehyde dehydrogenase and alcohol dehydrogenase activities. EhADH2 is the only known eukaryotic member of a newly defined family of prokaryotic multifunctional enzymes, which includes the Escherichia coli AdhE enzyme, an enzyme required for anaerobic growth of E. coli. Because of the critical role of EhADH2 in the amebic fermentation pathway and the lack of known eukaryotic homologues of the EhADH2 enzyme, EhADH2 represents a potential target for antiamebic chemotherapy. However, screening of compounds for antiamebic activity is hampered by the cost of large scale growth of Ent. histolytica, and difficulties in quantitating drug efficacy in vitro. To approach this problem, we expressed the EhADH2 gene in a mutant strain of E. coli carrying a deletion of the adhE gene. Expression of EhADH2 restored the ability of the mutant E. coli strain to grow under anaerobic conditions. By screening compounds for the ability to inhibit the anaerobic growth of the E. coli/EhADH2 strain, we have developed a rapid assay for identifying compounds with anti-EhADH2 activity. Using bacteria to bypass the need for parasite culture in the initial screening process for anti-parasitic agents could greatly simplify and reduce the cost of identifying new therapeutic agents effective against parasitic diseases.