CONTROL OF LEISHMANIA BY PROGRAMMED CELL DEATH (APOPTOSI

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The Leishmania parasite causes human disease with clinical symptoms ranging from-self healing cutaneous lesions to a fatal visceral infection. Additionally, in endemic areas, people infected with HIV are especially prone to Leishmania infection. The lack of understanding of cell biology and pathogenic mechanisms of this parasite makes the task of controlling this grave, worldwide health risk difficult. Closer to home it is particularly of concern to U.S. military personnel, their families and other travellers visiting or living in the endemic areas. To find novel methods for control of this pathogen,we have initiated study to understand the mechanism of programmed cell death (apoptosis) in Leishmania. Programmed Cell Death (PCD) is a phenomenon previously associated exclusively with multicellular organisms to regulate growth and development. It has been argued that unicellular organisms such as trypanosomatids, in order to promote and maintain genetic stability within the population, should get rid of unfit cells via a pathway similar to PCD. In the present study we have demonstrated that PCD exists in a unicellular protozoan parasite Leishmania donovani, a causative agent of visceral leishmaniasis. Two major developmental forms (promastigote and amastigote) are involved in the Leishmania life cycle. We report that PCD is initiated in both promastigotes and axenic amastigotes when they reach stationary phase in culture. In addition, PCD can be induced in these cells by drugs, pentavalent antimony [Pentostam] and amphotericin B, which are standard treatments for human visceral leishmaniasis. However, the treatment with these two drugs revealed differences in the induction of PCD in promastigotes and axenic amastigotes. The PCD in stationary phase and drug-treated cells is associated with nuclear condensation, DNA ladder formation and positive TUNEL assay. Using a substrate containing the tetra-peptide DEVD, the cleavage site for caspase 3-like proteases, we demonstrated that a caspase-like activity is induced at the time of PCD induction. To our surprise cycloheximide, a known inducer of cell death in certain higher eukaroytic cells, suppressed the induction of cell death in promastigotes and not in axenic amastigotes as judged by the lack of caspase-like activity and absence of DNA ladder. On the other hand antibiotic drug, geneticin (G418) commonly used to induce cell death in mammalian cells induced PCD in axenic amastigotes and was not able to significantly induce PCD in promastigotes. Thereby, suggesting that the two cell types of Leishmania respond differently to cell death signals and could present a challenge to treat this disease. Studies are on going to understand the mechanism of PCD induction and to characterize factors that are involved in the PCD pathway in unicellular organism such as Leishmania.