Caracterização molecular e bioquímica de Leishmania (Sauroleishmania) tarentolae: uma espécie não-patogênica

Abstract

Leishmania tarentolae is a species incapable of causing disease, even in severely immunocompromised individuals. This species infects reptiles and represents an important model for studying the biology of the genus Leishmania, since it shares a common ancestor with the pathogenic species. L. tarentolae has high biotechnological potential and has been widely used in the production of recombinant proteins and vaccines. Though several aspects regarding its own biology are unknown, including the presence of LPG, protease activity, sensitivity to complement and whether metacyclic forms are formed. Thus, the aim of the present study was to characterize biological aspects of L. tarentolae, using molecular and biochemical methodologies. The strains used in this study were L. tarentolae LV-414, L. amazonensis PH8, L. major Friedlin and L. braziliensis M2904. Metacyclogenesis in L. tarentolae was evaluated by comparing promastigotes in logarithmic and stationary phase by flow cytometry and by assessing the occurrence of changes in the composition of membrane carbohydrates by agglutination with lectins. Only a little percentage of small cells could be identified and their amount remained constant throughout the culture. The lectins from Concanavalia ensiformis and Triticum vulgaris induced same percentage of agglutination of logarithmic and stationary promastigotes. The presence of LPG in these parasites was evaluated using total protein extract and purified extract by western blotting using CA7AE and WIC 79.3 monoclonal antibodies. No antibody reactivity against purified extract was visualized, yet a band between 100-130 kDa was detected in the total protein extract. Disappearance of the bands after treatment with proteinase K confirmed the interaction of glycosylated proteins with CA7AE antibody. The profile of intracellular and secreted protease of L. tarentolae was analyzed by zymography assay. No proteases were detected in this species under the tested conditions, as opposed to L. amazonensis, which showed proteolytic activity both in the protein extract and culture supernatant. The sensitivity of L. tarentolae to the human complement system was evaluated in comparison to L. infantum. L. tarentolae was highly susceptible to complement in logarithmic and stationary phases of growth. The metabolic profile of L. tarentolae in the stationary phase was analyzed by GC-M and marked differences were seen among the profile of metabolites of the non-pathogenic and the pathogenic species, exhibiting lower abundance and/or absence of several metabolites related to the biosynthesis pathways of purines, pyrimidines, pentoses-phosphate pathway, glycolysis and the tricarboxylic acid cycle. This study contributed to the characterization of several aspects of the biology and metabolism of L. tarentolae, improving the application of these parasites as a model for studies of functional genomics, identification of virulence factors, development of vaccines and therapeutic targets.