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

BARROSO PAOLA ANDREA; AGUSTIN MOYA ALVAREZ; BRACAMONTE MARIA ESTEFANIA; HOYOS CARLOS LORENZO; ACUÑA LEONARDO; MARCO JORGE DIEGO

Parana, Entre Rios

Congreso; 54th Annual Meeting Agentine Society for Biochemistry and Molecular Biology LIV Reunion Anual Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2018

Sociedad Argentina de Investigación en 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, timeconsuming, and do no support automation for the parasite load quantification. The objective of this work was to standardize and validate amethod based on parasites expressing tomato red fluorescent protein in order to quantify the parasite load in an animal model of CL.The tomatogene was subcloned into pIR1SAT plasmid, and before the electroporation of Leishmania (Leishmania) amazonensis (MHOM/BR/73/M2269), itwas linearized with SwaI. The plasmid replaces one copy of the SSU rRNA gene, and the integration into Leishmania genome was confirmed byPCR. Parasites were selected in presence of nourseothricin and cloned in blood agar plate. Fluorescence was measured in intracellularamastigotes (am) obtained from cutaneous lesions of BALB/c mice in a plate reader. In addition, the infectivity of fluorescent parasites wascompared with the wild ones. After that, the efficacy of a topical treatment with epigallocatechin gallate (EGCg) in mice infected withLeishmania (L.) amazonensis was determined with the method standardized and compared with the conventional technique. The positive controlgroup was treated with of meglumine antimoniate.An excellent linear correlation was observed between the number of am and the fluorescenceemitted by the parasites (r2 = 0.98). In vivo, the parasites fluorescence was stable after several months post-infection, and the parasites wereinfective as the wild type. No difference was observed in the parasite load quantified by the fluorescence method and the conventional one. Onthe other hand, EGCg showed leishmanicidal activity inhibiting the parasite load in lesion (64 %).The fluorescence emitted by the tomato redprotein in transgenic parasites is a good indicator of parasites viability. The method was reproducible, cheap and useful for studying the efficacyof new leishmanicidal drugs in an animal model of cutaneous leishmaniasis.