Avaliação da manutenção da resistência ao dano no DNA da Leishmania amazonensis superexpressora do gene lamap armazenada a - 80°C

Abstract

Protozoa of the genus Leishmania are etiological agents of Leishmaniasis, belonging to the main Neglected Tropical Diseases and Endemic in Brazil. For the intracellular microorganisms elimination, such as Leishmania spp, immune cells produce reactive oxygen species (ROS) leading to damage to the parasite's DNA with the formation of AP abasic sites (apurinic/apyrimidine). Parasites such as Leishmania have DNA repair mechanisms, including the synthesis of AP Endonuclease enzymes. Lamap is an AP Endonuclease gene from Leishmania amazonensis, considered an important virulence factor, associated with the parasite's ability to survive oxidative stress and a possible therapeutic target. The main goal of this work was to evaluate whether the strain of L. amazonensis overexpressing the lamap gene cryopreserved at -80°C for 3 years would maintain cell viability against oxidative damage. In the Leishmania (L.) amazonensis response assay against the harmful agent menadione, it was observed that wild strain (JOSEFA) and the overexpressing lamap gene strain (JOSEFA LAMAP) showed reduced cell viability at 100μM menadione concentration (p < 0.0001). PCR confirmed the plasmid loss of the transfected strain. This plasmid loss may be associated with changes in the storage temperature of the strain stock, in addition to changing the antibiotic in the culture medium. The results of this work show the importance of controlling variables in a study. Processes such as storage, maintenance and manipulation of cells must have careful methodologies, as they are critical factors, especially at the molecular level.