Oxidative Stress-Induced Cell Death in Crithidia Fasciculata

Though cell death pathways similar to apoptosis have been observed in unicellular organisms, their mechanisms are yet to be elucidated. Crithidia fasciculata is a protozoan parasite that possesses a protein (CfB1-I) similar to anti-apoptotic BAX-1 inhibitor. To our knowledge, a BAX protein has not yet been discovered in trypanosomatids, raising the question of why these organisms might possess an anti-apoptotic protein with no clear binding partner. To lay the groundwork for exploring the role of CfB1-I in cell death, we are establishing a method to reliably induce cell death with oxidative stress and measure cellular responses. We hypothesize that hydrogen peroxide will induce cell death in C. fasciculata, as measured by fluorescence of acridine orange/propidium iodide. We treated wild type C. fasciculata with 0 - 5 mM hydrogen peroxide and measured the number of dead cells using propidium iodide using a microplate fluorometer and fluorescence microscopy. While initial results were inconclusive and suggest that C. fasciculata may be resistant to hydrogen peroxide, it is more likely that experimental conditions (e.g., exposure time, peroxide concentration, intensity of fluorescence from propidium iodide) need to be optimized to observe effects. Ultimately, we anticipate comparing sensitivity to oxidative stress in wild type cells compared to a cell line overexpressing CfB1-I.