Ethanol is the major metabolic product of glucose fermentation by the protozoan parasite Entmoeba histolytica under the anaerobic conditions found in the lumen of the colon. Here an internal peptide sequence determined from a major 39-kDa amoeba protein isolated by isoeLtric focusing followed by SDS/PAGE was used to clone the gene for the E. histolytica NADP+-dependent alcohol dehydrogenase (EhADH1; EC 1.1.1.2). The EhADHi clone had an open reading frame that was 360 amino acids long and encoded a protein of -39 kDa (calculated size). EhADHi showed a 62% amino acid identity with the tetrameric NADP+-dependent alcohol dehydrogenase of Thermoanaerobium brockii. In contrast, EhADHi showed a 15% amino acid identity with the closest human alcohol dehydrogenase. EhADHi contained 18 of the 22 amino acids conserved in other alcohol dehydrogenases, including glycines involved in binding NAD(P)+ as well as histidine and cysteine residues involved in binding the catalytic zinc ion. Like the T. brockii alcohol dehydrogenase, EhADHi lacked a 23-amino acid stretch present in other alcohol dehydrogenases that indudes four cysteines that bind a second noncatalytic zinc ion. An EhADH1-glutathione-Stransferase fusion protein showed the expected NADP+-dependent alcohol dehydrogenase and NADPH-dependent acetaldehyde reductase activities. The enzymatic activities of the EhADHi fusion protein were inhibited by pyrazole and 4-methylpyrazole.Entamoeba histolytica is a protozoan parasite that infects -500 million individuals in the developing world, resulting in -40 million cases of dysentery with a consequent annual mortality rate of ==40,000 (1, 2). Entamoebae reproduce under anaerobic conditions in the colonic lumen and cause dysentery when they invade the bowel wall. Because poor countries cannot afford to make the changes in sanitary conditions that might eliminate fecal-oral spread of amoebic infection, amebiasis is primarily controlled by drug treatment of persons sick with the parasite (1-3).Entamoebae are obligate fermenters because they lack mitochondria and the enzymes involved in respiration (4, 5). Amoebae use facilitated diffusion for the uptake of glucose, which is then metabolized to pyruvate via the Embden-

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Entamoeba histolytica causes amebic colitis and liver abscess in developing countries such as Mexico and India.Entamoeba dispar is morphologically identical but is not associated with disease. Here we determined the ploidy ofE. histolytica and developed PCR-based methods for distinguishing field isolates of E. histolytica or E. dispar. Fluorescence in situ hybridization showed that E. histolytica trophozoites are diploid for five “single-copy” probes tested. Intergenic sequences between superoxide dismutase and actin 3 genes of clinical isolates ofE. histolytica from the New and Old Worlds were identical, as were those of E. dispar. These results suggest a bottleneck or demographic sweep in entamoebae which infect humans. In contrast, E. histolytica and E. dispar genes encoding repeat antigens on the surface of trophozoites (Ser-rich protein) or encysting parasites (chitinase) were highly polymorphic.chitinase alleles suggested that the early axenized strains of E. histolytica, HM-1 from Mexico City, Mexico, and NIH-200 from Calcutta, India, are still present and that similarE. dispar parasites can be identified in both the New and Old Worlds. Ser-rich protein alleles, which suggested the presence of the HM-1 strain in Mexico City, included some E. histolytica genes that predicted Ser-rich proteins with very few repeats. These results, which suggest diversifying selection atchitinase and Ser-rich protein loci, demonstrate the usefulness of these alleles for distinguishing clinical isolates of E. histolytica and E. dispar.

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