E. histolytica is the protozoan parasite that causes 50 million cases of dysentery and liver abscess each year in the developing world. E. histolytica is primarily controlled by drug treatment of individuals sick with the parasite, because poor countries cannot afford to make the changes in sanitary conditions that might eliminate fecal-oral spread of amebic infection. E. histolytica trophozoites, which have no mitochondria, ferment pyruvate to ethanol under the anaerobic conditions in the colonic lumen. The amebic proteins involved in this fermentation, which are similar to those found in bacteria and unlike those found in man, are likely ancient and selected by anaerobic growth. Metronidazole, the only very effective drug against Entamoebae, targets the pyruvate:ferredoxin oxidoreductase and ferredoxin at the beginning of the amebic fermentation pathway. The proposed studies focus on amebic alcohol dehydrogenases (ADHs) at the end of the fermentation pathway. The amebic EhADH1 gene, which was cloned in our lab, encodes a 39-kDA zinc- and NADP+ dependent ADH (EC 1.1.1.2), which shows a remarkable resemblance to that of Thermoanaerobium brockii in its primary structure, substrate- and co-factor- specificities. EhADH1 is dissimilar to six human zinc-dependent ADHs in its primary structure, substrate- specificities, and co-factor requirements. Micromolar concentrations of pyrazole inhibited the EhADH1 enzyme, suggesting that EhADH1 may be a good target for anti-amebic drugs. The amebic EhADH2/EhALDH2 gene, which was serendipitously cloned elsewhere and closely resembled the E. coli ADHE gene, encodes a 96-kDa fusion enzyme composed of a putative iron- and NAD+-dependent ADH (EhADH2; EC 1.1.1.1) at the COOH-terminus and a CoA-dependent aldehyde dehydrogenase (EhALDH2; EC no 1.2.1.10) at the NH3-terminus.
In Specific Aim 1 effects of EhADH1 point mutations on zinc- and NADP+ binding are determined, ADH-inhibitors are tested versus EhADH1, and crystallization of EhADH1 is attempted;
In Specific Aim 2 EhADH1 and EhADH2 expression by amoebae under anaerobic conditions (model for the colonic lumen) and microaerophilic conditions (model for tissue invasion) is measured.
In Specific Aim 3 ADH-inhibitors are tested against amoebae growing in anaerobic and microaerophilic culture.
In Specific Aim 4 substrate and inhibitor specificities of EhADH2 are determined.
In Specific Aim 5 the phylogeny of the parasite fermentation enzymes is studied.
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