RESUMO
The 70 kDa heat shock protein (HSP70) is a molecular chaperone that assists the parasite Leishmania in returning to homeostasis after being subjected to different types of stress during its life cycle. In the present study, we evaluated the effects of HSP70 transfection of L. amazonensis promastigotes (pTEX-HSP70) in terms of morphology, resistance, infectivity and mitochondrial bioenergetics. The pTEX-HSP70 promastigotes showed no ultrastructural morphological changes compared to control parasites. Interestingly, the pTEX-HSP70 promastigotes are resistant to heat shock, H2O2-induced oxidative stress and hyperbaric environments. Regarding the bioenergetics parameters, the pTEX-HSP70 parasites had higher respiratory rates and released less H2O2 than the control parasites. Nevertheless, the infectivity capacity of the parasites did not change, as verified by the infection of murine peritoneal macrophages and human macrophages, as well as the infection of BALB/c mice. Together, these results indicate that the overexpression of HSP70 protects L. amazonensis from stress, but does not interfere with its infective capacity.
Assuntos
Animais , Feminino , Proteínas de Choque Térmico HSP70/fisiologia , Leishmania mexicana/fisiologia , Leishmaniose Cutânea/parasitologia , Proteínas de Protozoários/fisiologia , Estresse Fisiológico , Proteínas de Choque Térmico HSP70/genética , Leishmania mexicana/genética , Leishmania mexicana/ultraestrutura , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/fisiologia , Estresse Oxidativo , Proteínas de Protozoários/genética , Transfecção/métodosRESUMO
The 70 kDa heat shock protein (HSP70) is a molecular chaperone that assists the parasite Leishmania in returning to homeostasis after being subjected to different types of stress during its life cycle. In the present study, we evaluated the effects of HSP70 transfection of L. amazonensis promastigotes (pTEX-HSP70) in terms of morphology, resistance, infectivity and mitochondrial bioenergetics. The pTEX-HSP70 promastigotes showed no ultrastructural morphological changes compared to control parasites. Interestingly, the pTEX-HSP70 promastigotes are resistant to heat shock, H2O2-induced oxidative stress and hyperbaric environments. Regarding the bioenergetics parameters, the pTEX-HSP70 parasites had higher respiratory rates and released less H2O2 than the control parasites. Nevertheless, the infectivity capacity of the parasites did not change, as verified by the infection of murine peritoneal macrophages and human macrophages, as well as the infection of BALB/c mice. Together, these results indicate that the overexpression of HSP70 protects L. amazonensis from stress, but does not interfere with its infective capacity.
Assuntos
Proteínas de Choque Térmico HSP70/fisiologia , Leishmania mexicana/fisiologia , Leishmaniose Cutânea/parasitologia , Proteínas de Protozoários/fisiologia , Estresse Fisiológico , Animais , Feminino , Proteínas de Choque Térmico HSP70/genética , Leishmania mexicana/genética , Leishmania mexicana/ultraestrutura , Macrófagos/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Mitocôndrias/fisiologia , Estresse Oxidativo , Proteínas de Protozoários/genética , Transfecção/métodosRESUMO
Various Leishmania species were engineered with green fluorescent protein (GFP) using episomal vectors that encoded an antibiotic resistance gene, such as aminoglycoside geneticin sulphate (G418). Most reports of GFP-Leishmania have used the flagellated extracellular promastigote, the stage of parasite detected in the midgut of the sandfly vector; fewer studies have been performed with amastigotes, the stage of parasite detected in mammals. In this study, comparisons were made regarding the efficiency for in vitro G418 selection of GFP-Leishmania amazonensis promastigotes and amastigotes and the use of in vivo G418 selection. The GFP-promastigotes retained episomal plasmid for a prolonged period and G418 treatment was necessary and efficient for in vitro selection. In contrast, GFP-amastigotes showed low retention of the episomal plasmid in the absence of G418 selection and low sensitivity to antibiotics in vitro. The use of protocols for G418 selection using infected BALB/c mice also indicated low sensitivity to antibiotics against amastigotes in cutaneous lesions.