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
The natural biflavonoids morelloflavone-4â´-O-ß-D-glycosyl (1), (±)-fukugiside (2) and morelloflavone (3) were isolated from the ethyl acetate extract (EAEE) of dried and powdered fruit epicarps of Garcinia brasiliensis and derivatives of morelloflavone were semi-synthesised. Morelloflavone-7,4',7â³,3â´,4â´-penta-O-acetyl (4), morelloflavone-7,4',7â³,3â´,4â´-penta-O-methyl (5) and morelloflavone-7,4',7â³,3â´,4â´-penta-O-butanoyl (6) were prepared by acylation and alkylation reactions. All compounds showed leishmanicidal, antiproteolytic and antioxidant activities in addition to exhibiting low cytotoxicity. Compounds 4, 5 and 6 were highly active against Leishmania amazonensis promastigote forms compared to natural compounds of low lipophilicity, exhibiting IC(50) values of 0.0147, 0.0403 and 0.0189 µM, respectively. Compounds 4, 5 and 6 were also highly active against amastigote forms with IC(50) values of 0.042, 0.0603 and 0.059 µM, respectively. In addition, highly inhibitory activity against r-CPB2.8 and r-CPB3 isoforms was observed with these compounds. Notably, compounds 3 and 4 were the most active against r-CPB2.8 with IC(50) values of 0.4200 and 0.6744 µM, respectively. Compounds 5 and 6 also showed significant inhibitory activities with very similar IC(50) values of 1.2663 and 1.0122, respectively. However, compounds 1 and 2 exhibited the lowest inhibitory activity against r-CPB2.8, almost 6 and 11-fold less active than the natural compound 3. In L. (L.) amazonensis lysates, and compounds 3 and 6 were the most active inhibitors of amastigote lysates at pH 5, which is near the pH environment of the parasitophorous vacuole within the macrophage. Finally, compounds 1, 2 and 3 exhibited effective antioxidant activity compared to the reference antioxidant ascorbic acid. However, the activity was lower than that of butylhydroxytoluene (BHT), which may be related to the reduced number of phenolic hydroxyl groups that were replaced by more lipophilic substituents in derivatives 4-6. Compounds 4-6 exhibited reduced antioxidant activity as evidenced by their higher EC(50) values. These results provide new perspectives on drug development for the treatment of leishmaniasis and inhibitory enzyme activity on Leishmania (L.) mexicana cysteine proteases and other isoforms.
