Abstract
Antimonials continue to be considered the first-line treatment for leishmaniases, but its use entails a wide range of side effects and serious reactions. The search of new drugs requires the development of methods more sensitive and faster than the conventional ones. We developed and validated a fluorescence assay based in the expression of tdTomato protein by Leishmania, and we applied this method to evaluate the activity in vitro of flavonoids and reference drugs. The pIR1SAT/tdTomato was constructed and integrated into the genome of Leishmania (Leishmania) amazonensis. Parasites were selected with nourseothricin (NTC). The relation of L. amaz/tc3 fluorescence and the number of parasites was determined; then the growth in vitro and infectivity in BALB/c mice was characterized. To validate the fluorescence assay, the efficacy of miltefosine and meglumine antimoniate was compared with the conventional methods. After that, the method was used to assess in vitro the activity of flavonoids; and the mechanism of action of the most active compound was evaluated by transmission electron microscopy and ELISA. A linear correlation was observed between the emission of fluorescence of L. amaz/tc3 and the number of parasites (r2 = 0.98), and the fluorescence was stable in the absence of NTC. No differences were observed in terms of infectivity between L. amaz/tc3 and wild strain. The efficacy of miltefosine and meglumine antimoniate determined by the fluorescence assay and the microscopic test showed no differences, however, in vivo the fluorescence assay was more sensitive than limiting dilution assay. Screening assay revealed that the flavonoid galangin (GAL) was the most active compound with IC50 values of 53.09 µM and 20.59 µM in promastigotes and intracellular amastigotes, respectively. Furthermore, GAL induced mitochondrial swelling, lipid inclusion bodies and vacuolization in promastigotes; and up-modulated the production of IL-12 p70 in infected macrophages. The fluorescence assay is a useful tool to assess the anti-leishmanial activity of new compounds. However, the assay has some limitations in the macrophage-amastigote model that might be related with an interfere of flavanol aglycones with the fluorescence readout of tdTomato. Finally, GAL is a promising candidate for the development of new treatment against the leishmaniasis.
Highlights
The leishmaniases are a group of diseases caused by parasites of the genus Leishmania, which are transmitted to humans through the bite of infected female phlebotomine sandflies
The aims of this work were to develop and validate a fluorescence assay based in the expression of tdTomato protein by Leishmania (L.) amazonensis, and to apply this method to evaluate in vitro the activity of flavonoids and reference drugs
The pIR1SAT-tdTomato plasmid was linearized with SwaI restriction enzyme before transfection of L. (L.) amazonensis parasites (Figure 1A)
Summary
The leishmaniases are a group of diseases caused by parasites of the genus Leishmania, which are transmitted to humans through the bite of infected female phlebotomine sandflies These diseases in all their clinical expressions are endemic in 98 countries (Alvar et al, 2012), affecting mainly low income populations of the tropical and subtropical belt worldwide. Reporter gene (RG) technology has become one of the most promissory and widely used tool for drug screening in several models since it offers live imaging, high sensibility, specificity and flexibility (Pulido et al, 2012) These genes typically encode a gene product that has a readily measurable phenotype and is distinguishable over endogenous cellular background (Dube et al, 2009). The choice of a RG depends on the cell line used, the nature of the experiment, and the adaptability of the assay to the appropriate detection method (New et al, 2003)
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