This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Anaerobic pathogens have acquired traits to survive and thrive in host guts. As a model, we study the causative agent of human amebiasis, Entamoeba histolytica. This disease affects 50 million people yearly with 100 thousand fatalities worldwide. Entamoeba histolytica's life cycle consists of the disease-causing trophozoite stage and the infectious cyst stage. Cysts are acquired by ingestion of contaminated food or water, and multiplication and differentiation of the emergent trophozoites happen within the human's large intestine. Entamoeba histolytica lacks mitochondria and obtains its energy from the fermentation of glucose, producing carbon dioxide, acetate, and ethanol. The bifunctional Entamoeba histolytica alcohol dehydrogenase 2 (EhADH2) is essential for the growth and survival of E. histolytica trophozoites. That their evolutionary origin apparently differs from that of vertebrate enzymes designates them as appealing antimicrobial targets. The goal of this study is to search for alternative non-toxic drugs that inhibit the growth of E. histolytica trophozoites, by specifically affecting the EhADH2 enzyme. Animal models will be used to determine the toxicity of these drugs in humans.
Three specific aims will help achieve this goal: (1) Identify candidate chemicals that inhibit EhADH2. First, test potential inhibitors in vitro using an Escherichia coli AdhE deficient strain, and later in vivo using Entamoeba histolytica trophozoites. Useful candidates should inhibit the enzymatic activity of EhADH2, killing trophozoites with little or no effect to mammalian enzymes. (2) The selected chemicals will be further analyzed in cell cultures. To evaluate the toxicity of these drugs, various concentrations of drugs will be tested in animal models (SCID mice). LD50 standard procedures will be used. (3) Further analysis of EhADH2 will aid in designing anti-amoebic agents. EhADH2 (wild-type, mutants) will be analyzed with identified and designed inhibitors, using classical kinetics, spectrophotometer, computer modeling, and x-ray crystallography techniques.
Showing the most recent 10 out of 376 publications
