Abstract 1719 Polyamine Depletion Induces an Apoptotic-like Death Phenotype in Leishmania Parasites

Leishmaniasis is on the list of Top Ten neglected tropical diseases in the World and is most common in developing countries. The disease is caused by the protozoan parasites Leishmania spp., which are transmitted to the mammalian host by the bite of a female sand fly. There is no vaccine, and the currently available limited number of medications have side effects, are expensive, and drug resistance is emerging. Thus, there is an urgent need for new therapeutic targets and recent research has emphasized the importance of polyamines for Leishmania parasites. Our overall goal is to discover the functions of polyamines for parasite growth and survival and my research focused on the effect of polyamine depletion on cell death. The main polyamine biosynthetic enzymes in Leishmania are ornithine decarboxylase (ODC) and spermidine synthase (SPDSYN), which sequentially convert ornithine to the polyamines, putrescine and spermidine. We utilized two gene deletion mutants, Δodc and Δspdsyn, to determine the effect of putrescine and spermidine depletion in Leishmania donovani. We hypothesize that polyamine depletion causes an apoptosis-like cell death and investigated two hallmarks of apoptosis: DNA fragmentation and membrane modification. We used a TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay to detect DNA fragmentation and annexin/propidium iodide staining to detect membrane modification. The TUNEL assay is based on detecting free 3 hydroxyl-ends that are generated by endonuclease digestion during apoptosis. Double staining with annexin and PI is commonly used to detect apoptotic cells: annexin labels the exposed membrane phospholipid phosphatidylserine while PI does not enter live or apoptotic cells but does enter necrotic cells. Analysis via flow cytometry revealed that polyamine-depleted Δodc and Δspdsyn mutants showed significantly more DNA fragmentation and membrane modification than wild type parasites or polyamine-supplemented mutants. Interestingly, the Δodc mutants exhibited even more profound signs of DNA fragmentation and membrane modification compared to the Δspdsyn mutants, suggesting that putrescine depletion triggers an apoptosis-like death phenotype in Leishmania parasites. In conclusion, our studies validate the polyamine biosynthetic pathway, especially the enzyme ODC, as a promising therapeutic target. Research reported in this poster was supported by NIAID of the National Institutes of Health under award number R15AI151980.