Development of Leishmania (Leishmania) Amazonensis expressing fluorescent tomato red protein for in vitro and in vivo screening of anti-leishmanial drugs

PEREZ BRANDAN, MC; HOYOS, CARLOS L; BARROSO PA; PORTELLI S. N.; BRACAMONTE M E; BASOMBRÍO, MIGUEL A; MARCO, J DIEGO

Toledo

Congreso; 6th World Congress on Leishmaniasis; 2017

Worldleish 6

Background. The evaluation of anti-leishmanial activity of novel drugs is based on methods which are laborious, time consuming, and do no support automation for the parasite load quantification. Recently, many reporter molecules such as green fluorescent protein, B-lactamase and luciferase have been devised for thequantification of parasites with different degrees of sensitivity. The objective of this work was to standardize and validate a method based on parasites expressing tomato red fluorescent protein, applicable not only for in vitro parasite quantification, but also for in vivo experiments. Methods. The tomato gene was subcloned into pIR1SAT plasmid, and before the electroporation of Leishmania (Leishmania) amazonensis, it was linearized with SwaI. The plasmid replaces one copy of the SSU rRNA gene, and the integration into Leishmania genome was confirmed by PCR. Parasites were selected in presence of nourseothricin and cloned in blood agar plate. Fluorescence was measured in promastigotes (pm), intracellular amastigotes (am) in macrophage cultures as well as in am obtained from cutaneous lesions of BALB/c mice in a plate reader. In addition, the infectivity of fluorescent parasites was compared with the wild ones. The leishmanicidal activity of miltefosine (IC50) in vitro and the efficacy of meglumine antimoniate (120mg Sbv/kg/day) during 20 days were determined with the method standardized and compared with the conventional techniques. Results. An excellent linear correlation was observed between the number of pm or am and the fluorescence emitted by the parasites (r2 = 0.98). In vitro, the values of IC50 of miltefosine against pm and am assessed either measuring the fluorescence or by counting the parasites with an optical microscope were similar between themethods. In vivo, the fluorescence of parasites was stable during the infection in mice and the treatment with meglumine antimoniate inhibited 48% to 55% of the load parasite in lesions. Conclusions. The fluorescence emitted by the tomato red protein in transgenic parasites is a good indicator of parasites viability, useful for the screening of anti-leishmanial drugs in a simple and fast way both in vitro and in vivo model of Leishmania (L.) amazonensis.