RESUMO
Leishmania parasites cause leishmaniasis, one of the most epidemiologically important neglected tropical diseases. Leishmania exhibits a high ability of developing drug resistance, and drug resistance is one of the main threats to public health, as it is associated with increased incidence, mortality, and healthcare costs. The antimonial drug is the main historically implemented drug for leishmaniasis. Nevertheless, even though antimony resistance has been widely documented, the mechanisms involved are not completely understood. In this study, we aimed to identify potential metabolite biomarkers of antimony resistance that could improve leishmaniasis treatment. Here, using L. tropica promastigotes as the biological model, we showed that the level of response to antimony can be potentially predicted using 1H-NMR-based metabolomic profiling. Antimony-resistant parasites exhibited differences in metabolite composition at the intracellular and extracellular levels, suggesting that a metabolic remodeling is required to combat the drug. Simple and time-saving exometabolomic analysis can be efficiently used for the differentiation of sensitive and resistant parasites. Our findings suggest that changes in metabolite composition are associated with an optimized response to the osmotic/oxidative stress and a rearrangement of carbon-energy metabolism. The activation of energy metabolism can be linked to the high energy requirement during the antioxidant stress response. We also found that metabolites such as proline and lactate change linearly with the level of resistance to antimony, showing a close relationship with the parasite's efficiency of drug resistance. A list of potential metabolite biomarkers is described and discussed.
Assuntos
Antimônio/toxicidade , Antiprotozoários/toxicidade , Resistência a Medicamentos , Leishmania tropica/metabolismo , Metaboloma , Metabolismo Energético , Leishmania tropica/efeitos dos fármacos , Pressão Osmótica , Estresse OxidativoRESUMO
ATPase activity has been located on the external surface of Leishmania tropica. Since Leishmania is known to have an ecto-acid phosphatase, in order to discard the possibility that the ATP hydrolysis observed was due to the acid phosphatase activity, the effect of pH in both activities was examined. In the pH range from 6.8 to 8.4, in which the cells were viable, the phosphatase activity decreased, while the ecto-ATPase activity increased. To confirm that the observed ATP hydrolysis was promoted by neither phosphatase nor 5'-nucleotidase activities, a few inhibitors for these enzymes were tested. Vanadate and NaF strongly inhibited the phosphatase activity; however, no effect was observed on ATPase activity. Neither levamizole nor tetramizole, two specific inhibitors of alkaline phosphatases, inhibited this activity. The lack of response to ammonium molybdate indicated that 5'-nucleotidase did not contribute to the ATP hydrolysis. Also, the lack of inhibition of the ATP hydrolysis by high concentrations of ADP at nonsaturating concentrations of ATP discarded the possibility of any ATP diphosphohydrolase activity. The ATPase here described was stimulated by MgCl2 but not by CaCl2. In the absence of divalent metal, a low level of ATP hydrolysis was observed, and CaCl2 varying from 0.1 to 10 mM did not increase the ATPase activity. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.29 +/- 0.02 mM MgCl2. The apparent K(m) for Mg-ATP2- was 0.13 +/- 0.01 mM and free Mg2+ did not increase the ATPase activity. ATP was the best substrate for this enzyme. Other nucleotides such as ITP, CTP, GTP, UTP, and ADP produced lower reaction rates. To confirm that this Mg-dependent ATPase was an ecto-ATPase, an impermeant inhibitor, 4,4'-diisothiocyanostylbene-2,2'-disulfonic acid was used. This amino/sulfhydryl-reactive reagent did inhibit the Mg-ecto-ATPase activity in a dose-dependent manner (I0.5 = 27.5 +/- 1.8 microM).
Assuntos
Adenosina Trifosfatases/metabolismo , Leishmania tropica/enzimologia , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , 4-Nitrofenilfosfatase/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Concentração de Íons de Hidrogênio , Leishmania tropica/metabolismo , Magnésio/farmacologia , Nitrofenóis/metabolismo , Compostos Organofosforados/metabolismo , Especificidade por SubstratoRESUMO
Metacyclic forms of Leishmania major and putative metacyclics of L. mexicana mexicana were found to occur in abundance in stationary phase cultures. These forms have been compared in several ways with promastigotes from mid-log phase cultures and, in the case of L. m. mexicana, amastigotes. Metacyclics are smaller, contain less protein and appear more active than other promastigotes. Both forms of promastigote respire at a high rate in the absence of exogenous substrate. The free amino-acid contents of the various forms of the two species have been analysed. They differ in detail but alanine was the major amino acid in all cases. The isoenzyme content of the different forms differed significantly. That of the putative metacyclics of L. m. mexicana was in several respects more similar to amastigotes than promastigotes, suggesting that the form is pre-adapted for life in a mammal. Metacyclics of L. major apparently did not divide in culture but transformed back over a period of 48 h to mid-log phase cells. The results provide further detail of the molecular differences between mid-log phase and metacyclic promastigotes and confirm that metacyclics are a distinct form in the life-cycle.