Fluorescent Leishmania parasites, a useful method for studying the efficacy of new drugs

BARROSO, PA; ALVAREZ MOYA, A; BRACAMONTE E; HOYOS, C; ACUÑA L; MARCO, JD

Paraná

Congreso; LIV reunión anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular.; 2018

Sociedad Argentina de Investigación Bioquímica y Biología Molecular.

The conventional methods for assessing the efficacy of a new drug in an animal model of cutaneous leishmaniasis (CL) are laborious, time consuming, and do no support automation for the parasite load quantification. The objective of this work was to standardize and validate a method based on parasites expressing tomato red fluorescent protein in order to quantify the parasite load in an animal model of CL.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 intracellular amastigotes (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. After that, the efficacy of a topical treatment with epigallocatechin gallate (EGCg) in mice infected with Leishmania (L.) amazonensis was determined with the method standardized and compared with the conventional technique. The positive control group was treated with of meglumine antimoniate.An excellent linear correlation was observed between the number of am and the fluorescence emitted by the parasites (r2 = 0.98). In vivo, the parasites fluorescence was stable after several months post-infection, and the parasites were infective as the wild type. No difference was observed in the parasite load quantified by the fluorescence method and the conventional one. On the other hand, EGCg showed leishmanicidal activity inhibiting the parasite load in lesion (64 %).The fluorescence emitted by the tomato red protein in transgenic parasites is a good indicator of parasites viability. The method was reproducible, cheap and useful for studying the efficacy of new leishmanicidal drugs in an animal model of cutaneous leishmaniasis.