Leishmania parasite causes human disease (Leishmaniasis) with clinical symptoms ranging from self healing cutaneous lesions to fatal visceral infection. The lack of understanding of the mechanism by which Leishmania parasite causes disease poses a serious public health risk worldwide and in particular for U.S. military personnel their families and tourists either living or travelling in endemic areas. As a first step towards understanding the molecular mechanism of Leishmania pathogenesis, we have began to analyze the processes that are involved in parasite life cycle in transformation from an avirulent to virulent form and to target such processes to control growth of the parasite. The role of unique structural and functional features of organelles, like the cytoskeleton and the flagellar apparatus, in infectivity of Leishmania, is still obscure. In order to identify genes that control growth, we have isolated for the first time a gene encoding for centrin from L. donovani. Centrin is calcium binding cytoskeletal protein essential for centrosome duplication or segregation. The levels of centrin mRNA and protein were high during the exponential growth of the parasite in culture and declined to a low level in the stationary phase. Expression of N-terminal deleted centrin in the parasite significantly reduces its growth rate and it was found that significantly more cells are arrested in the G2/M stage than in control cells. Centrin mutant parasites were shown to be highly susceptible to killing by the human macrophages suggesting growth arrested centrin mutants have an attenuated phenotype. Further, these studies indicate that centrin may have a functional role in Leishmania growth.Currently studies are underway to identify proteins which interact with centrin are important for its function. Modulation of expression of centrin interacting gene products would hopefully allow us to further attenuate the parasite growth and to generate a vaccine candidate. Programmed cell death (PCD) is an essential part of cell biology and is thought to have evolved not only to regulate growth and development in multicellular organisms. However, recent studies, which showed the existence of PCD in unicellular organisms, have postulated a functional role of PCD in the biology of unicellular organisms. It has been postulated that in order to promote and maintain clonality within the population, the Trypanosomatids must have developed an altruistic mechanism to control growth. We wanted to explore whether PCD exists in Leishmania and if so can antiLeishmanial drugs induce it. This will provide an opportunity to develop future antiLeishmanial drugs. We have demonstrated some features characterizing programmed cell death (PCD) in the unicellular protozoan parasite Leishmania donovani, the causative agent of visceral Leishmaniasis. We report that PCD is initiated in stationary phase cultures of promastigotes and both in actively growing cultures of axenic amastigotes and promastigotes upon treatment with anti Leishmanial drugs (Pentostam and amphotericin B). However, the two cell types respond to antiLeishmanial drugs differently. The features of PCD in Leishmania donovani promastigotes and amastigotes are nuclear condensation, nicked DNA in the nucleus, DNA ladder formation, increased plasma membrane permeability,decrease in mitchondrial membrane poential, and induction of casapase-like activity. Therefore, these studies provide for the first time the presence of programmed cell death in Leishmania and the basis for understanding the mechanism of Leishmania pathogenesis. Currently we are charctertizing the factors involved in the cell death program with an aim to exploite it for the control of parasite growth.
