Date of Award
Master of Science (MS)
Temesvari, Lesly A
Henson , J. Michael
Paul , Kimberly S
Amoebiasis is defined as an intestinal infection with the human protozoan parasite, Entamoeba histolytica; approximately 100,000 deaths annually can be attributed to amoebiasis. Disease states range from asymptomatic colonization to invasive amoebiasis, characterized by abdominal pain and dysentery. Dissemination to extraintestinal sites, such as the liver and/or brain, can occur. Vesicle trafficking is critical for parasite virulence, and multiple signaling cascades within the pathogen facilitate these events.
One important regulator of signal transduction is the tightly controlled PI 3-kinase (PI3K), which phosphorylates the lipid, phosphatidylinositol bisphosphate (PIP2), producing phosphatidylinositol trisphosphate (PIP3). Effector proteins, such as Akt, are recruited to the signaling complex, where they initiate downstream signal events. These effector proteins often possess lipid binding domains specific for PIP3, such as the Pleckstrin homology (PH) domain. In mammalian cells, PI3K signaling pathways play a role in the prevention of apoptosis, a type of programmed cell death (PCD). Like mammalian cells, several unicellular eukaryotic organisms, including E. histolytica, undergo PCD in response to stimuli such as exposure to reactive oxygen species or drugs. Interestingly, metronidazole (MTZ), the DNA damaging drug commonly used to treat E. histolytica infections, can induce PCD in another protozoan, Blastocystis hominis.
Recently, an E. histolytica cell line expressing GFP-labeled PHBTK was generated, which exhibited a PI3K dominant-negative phenotype (PI3K-DN), presumably because the binding of PHBTK to PIP3 prevents binding and signaling by physiologically relevant proteins. In the current study, we investigated the effectiveness of MTZ on the E. histolytica PI3K-DN cell line. Because at least one organism undergoes PCD in response to the DNA damaging drug, MTZ, and since PI3Ks are involved in cell survival signals in mammalian cells, we hypothesized that E. histolytica cells expressing GFP-PHBTK, putatively deficient in cell survival signals, may represent a cell line that is more susceptible to MTZ killing. We found that cells expressing GFP-PHBTK were resistant to MTZ killing, refuting our initial hypothesis.
Therefore, we investigated the connection between PI3K-DN signaling and resistance to MTZ. Wildtype cells treated with a chemical PI3K inhibitor displayed increased MTZ resistance. Known MTZ resistance mechanisms, including expression of multidrug resistant pumps and increased levels of superoxide dismutase, were not related to expression of GFP-PHBTK. Interestingly, we found that MTZ induced phenotypes associated with PCD in both wildtype E. histolytica cells and cells expressing GFP-PHBTK. This is the first report that demonstrates that inhibition of PI3Ks contributes to MTZ resistance in E. histolytica, and the first report that shows MTZ can induce PCD in the parasite.
Powell, Rhonda, "Inhibition of PI 3-kinase signaling contributes to metronidazole resistance in the protozoan parasite, Entamoeba histolytica" (2009). All Theses. 684.