Complementation of an Escherichia coli adhE mutant by the Entamoeba histolytica EhADH2 gene provides a method for the identification of new antiamebic drugs.
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.
Yong
T S
TS
Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
Li
E
E
Clark
D
D
Stanley
S L
SL
D000426:Alcohol Dehydrogenase / Q000235:genetics*; D000445:Aldehyde Oxidoreductases / Q000235:genetics*; D000818:Animals; D000981:Antiprotozoal Agents / Q000494:pharmacology*; D003001:Cloning, Molecular; D004748:Entamoeba histolytica / Q000187:drug effects / Q000235:genetics*; D029968:Escherichia coli Proteins; D005816:Genetic Complementation Test; D008826:Microbial Sensitivity Tests; D009097:Multienzyme Complexes / Q000235:genetics*; D009154:Mutation; D013379:Substrate Specificity; D014170:Transformation, Genetic
D000981:Antiprotozoal Agents; D029968:Escherichia coli Proteins; D009097:Multienzyme Complexes; D000426:Alcohol Dehydrogenase; C068383:adhE protein, E coli; D000445:Aldehyde Oxidoreductases