Effect of methyl ester of L-leucina on the proliferation and the ultrastructure of the Trypanosoma cruzi
L-amino acid methyl esters, such as L-leucine methyl ester (Leu-OMe), have been identified as targeting agents for amastigotes lysosomal system of the trypanosomatid Leishmania (Leishmania) amazonensis, by a mechanism that involves ester hydrolysis by parasite enzymes located in megasomes. Thus, in this study we analyzed the effect of Leu-OMe on all three evolutive forms of the trypanosomatid Trypanosoma cruzi searching for potential targets of the compound in this protozoan. Treatment of epimastigote forms resulted in dose-dependent growth inhibition, with IC50/1day = 0.55 ± 0.21 mM. Incubation with 4-8 mM/1day led to 100% cell death. Treatment of bloodstream trypomastigotes resulted in cell lysis, with an IC50/1day = 1.46 ± 0.16 mM. Furthermore, infected macrophages treated with 0.125 to 1 mM of Leu-OMe showed a dose- and time-dependent decrease in the percentage of amastigotes infection. Morphological changes in macrophages were observed only at concentrations above 8 mM on the third day of treatment. Analysis by transmission electron microscopy of treated parasites demonstrated severe morphological alterations in cell shape, mitochondria and nucleus, while kinetoplast and reservosomes (pre-lysosomal compartment) appeared not to be affected. Lysis of bloodstream trypomastigotes and intracellular amastigotes indicated that lysosomes of T. cruzi are the main target for the drug, since reservosomes are found only in epimastigote forms. Our results allowed to demonstrate, by using aryl sulphatase ultrastructural cytochemistry, the presence of lysosomes in T. cruzi epimastigotes, and thus confirmed the susceptibility of T. cruzi lysosomes to Leu-OMe, leading to parasite death. This cytochemical method was used for the first time in the trypanosomatid protozoa T. cruzi and Crithidia fasciculata, showing positive reaction exclusively inside membrane-bound cytoplasmic vacuoles distributed throughout the cell body. Furthermore, no positive reaction was detected in epimastigote reservosomes, compartments which are considered pre-lysosomal organelles. Thus, our data show that reservosomes and lysosomes are distinct organelles in T. cruzi epimastigotes. The positive reaction in cytoplasmic vesicles of C. fasciculata choanomastigotes confirmed the specificity of the reaction for lysosomes in other trypanosomatid species.
Advisor:Maurilio José Soares; Solange Lisboa de Castro
School:Faculdades Oswaldo Cruz
Source Type:Master's Thesis
Keywords:Trypanosoma cruzi Lisossomos Chagas Disease Drug Therapy Leucine Lysosomes methyl ester
Date of Publication:06/23/2006