Analysis Of The Different Stage By Using Serial Gene Expression And Effect HSP90 During Process Transformation In Leishmania Donovani

Leishmania is a protozoan parasite known to cause widespread human diseases around the world. Leishmaniasis is a major and increasingly prevalent public health problem in many regions of the world, particularly in Africa, Asia, and South America. These protozoan parasites have a life cycle characterized by the presence of a flagellated promastigote stage within the sand fly host and a nonmotile amastigote stage within the mammalian host. The promastigote-to-amastigote cytodifferentiation is a profound morphological and physiological transformation. During the process of differentiation, the parasite loses its flagellum, rounds up, changes its glycoconjugate coat, and begins to express a set of metabolic enzymes optimally active at a low pH. The transformation of Leishmania promastigotes to amastigotes during the infection of the host macrophage appears to involve a series of steps. These steps not only result in morphologic transformation, but also allow survival within the parasitophorous vacuole. The promastigote-amastigote cytodifferentiation's significance in establishing an infection within the mammalian host has prompted us to identify molecular events involved in this process.In this study, we examined the transcriptome of Leishmania donovani promastigotes and axenic amastigotes to identify differentially regulated mRNAs utilizing the serial analysis of gene expression (SAGE). The axenic culture of amastigotes was initiated from stationary-phase promastigotes. Transformation from promastigote to amastigote occurred when cultures in Medium 199 (pH 5.5), supplemented with 20% (v/v) FBS, were transferred from 26℃to 37℃. A total of 20,299 and 20,132 tags were generated from promastigote and amastigote libraries, respectively. The containing unique genes identified in these two SAGE libraries were 8,615 and 7,835, respectively.Characteristics of the expressed genes'frequency distribution were remarkably similar in both libraries: the most abundant tags (frequency≥20), whose levels were equal to or > 1.3% of the identified tags, constituted > 23% of the total sequenced tags. Correspondingly, 75.72%, or 71.65% of the genes accounted for those tags present at low abundance (frequency=1), contributed only 32.13%, or 27.89%, of the total tags. A total of 968 genes (11.2% of the total genes in promastigotes and 12.4% in amastigotes) were recorded to have statistically different transcript levels between promastigotes and axenic amastigotes. Of the 968 distinct total genes, there are 326 promastigote-enriched transcripts and 642 amastigote-enriched mRNAs. Additional confirmation of the SAGE data was obtained utilizing quantitative real-time PCR.The present study also investigates the role of geldanamycin (GA),a specific inhibitor of HSP90, during L. donovani promastigote-to- amastigote transformation stage in axenic conditions. Previous study demonstrated that promastigote-to-amastigote differentiation could be induced at a low temperature (25°C) and neutral pH by using GA. Curiously, another study has shown that heat stress triggers a process of programmed cell death in Leishmania infantum promastigotes. Hence, this prompted us to study the effects of GA and pH of media during the promastigote-to-amastigote transformation stage. Primary interest lies in knowing whether GA can induce apoptosis-like death or stage-transformation in L. donovani promastigote at a high temperature and a low pH. Moreover, finding a possible evidence of Hsp90 protection pathway is anticipated and the effects of media pH during GA treatment. In lieu of this, five selected gene expression levels between GA treated and untreated cells were also evaluated. These are selected from the previous results of serial analysis of gene expression (SAGE), which exhibited significantly different expression levels between L. donovani promastigote and amastigote stages. Promastigotes exhibited morphologic changes, including cell shrinkage, cell rounding, and cytoplasmic blebbing after GA treatment at a high temperature. The positive apoptosis cells could be observed in situ by TdT-dUTP terminal nick-end labeling (TUNEL). Flow cytometry analysis shows a significant increase (P<0.01) in proportion to apoptotic cells with the effect in a dose- and time-dependant manner. Meanwhile, cell cycle analysis with propidium iodide stain shows a significant increase in the G1/G0 phase and a decrease in the S and G2/M phases (P<0.05). In addition, cellular glutathione level was reduced and reactive oxygen species (ROS) was increased afterwards. Pretreatment with antioxidants bring down the percentage of GA induced cell apoptosis. After treatment, cultures in pH 5.5 showed a lower percentage of apoptosis than in pH 7.4(P<0.05), indicating that acidic environments with a high temperature may play a protective role during the transformation stage. In sum, this study provides further evidence that both the Hsp90 and acidic conditions are likely to be crucial to the transformation and survival of the parasite within its human host.