Date of Award
Master of Science (MS)
Lesly A Temesvari
James C Morris
Entamoeba histolytica is the causative agent of amebic dysentery and is prevalent in developing countries. It has a biphasic lifecycle: active, virulent trophozoites and dormant, environmentally-stable cysts. Only cysts are capable of establishing new infections, and are spread by fecal deposition. Many unknown factors influence stage conversion, and synchronous encystation of E. histolytica is not currently possible in vitro. E. histolytica infections are treated with nitroimidazole drugs, such as metronidazole (Flagyl™). However, several clinical isolates have shown metronidazole resistance. Enhancing the amebicidal mechanisms of metronidazole through drug combination therapy may allow for more effective treatment. Metronidazole may cause amebic death through double-stranded DNA breakage. Inhibiting homologous recombination in E. histolytica would prevent recovery from damage induced by metronidazole and may potentiate the action of this drug. Rad51 and Dmc1 are homologous DNA recombinases involved in double-stranded DNA repair that are expressed in E. histolytica. However, the physiological roles of these two proteins are unknown. Both Rad51 and Dmc1 are upregulated during encystation of Entamoeba invadens, an in vitro model organism. Additionally, Rad51 activity is decreased by small molecule inhibitors. To gain insight into the physiological roles of Rad51 and Dmc1 in E. histolytica, and to determine drug target potential, we developed two knockdown cell lines using the RNAi-mediated Trigger approach. We transfected healthy trophozoites with gene-specific Trigger plasmid constructs and verified knockdown status using RT-PCR and Western blots. Neither Rad51 nor Dmc1 was consistently knocked down in Trigger transfected cell lines. Because these knockdowns were potentially lethal to the cells, we also exposed healthy E. histolytica trophozoites to metronidazole combined with small molecule Rad51 inhibitors B02 and DIDS to determine if we could lower the IC50 of metronidazole. Interestingly, B02 was not cytotoxic and did not consistently alter the IC50 of metronidazole. DIDS was only cytotoxic at high concentrations, but lower concentrations seemed effective in decreasing the IC50 of metronidazole, suggesting that DIDS may be additive or synergistic. Overall, these data may suggest that combination therapy using B02 and metronidazole would not yield more effective treatment than standard metronidazole regimens. These data may also suggest that a combination of DIDS and metronidazole would yield a more effective amebiasis treatment.
Ham, Rachel Evelyn, "Investigating Rad51 and Dmc1 in Entamoeba histolytica: Homologous Recombinases with Drug Target Potential" (2020). All Theses. 3392.