RESUMO
Trypanosomatid parasites containing a metabolically unstable ornithine decarboxylase (ODC) are naturally resistant to high levels of alpha-difluoromethylornithine (DFMO) because this ODC inhibitor, though causing a drastic reduction of intracellular putrescine, elicits only a moderate decrease of the spermidine endogenous pool. In this study we have used a combination of DFMO with cyclohexylamine (CHA; bis-cyclohexylammonium sulfate), an inhibitor of spermidine synthase, to reach a more complete depletion of spermidine. Under these conditions we have observed the arrest of proliferation not only in trypanosomatids with stable ODC but also in parasites with an enzyme of high turnover rate. In all cases the reinitiation of proliferation occurred only after the addition of exogenous spermidine, and neither putrescine nor spermine were able to induce the same effect.
Assuntos
Crithidia fasciculata/crescimento & desenvolvimento , Espermidina/metabolismo , Trypanosoma cruzi/crescimento & desenvolvimento , Animais , Crithidia fasciculata/efeitos dos fármacos , Crithidia fasciculata/enzimologia , Crithidia fasciculata/metabolismo , Cicloexilaminas/farmacologia , Eflornitina/farmacologia , Inibidores Enzimáticos/farmacologia , Ornitina Descarboxilase/metabolismo , Putrescina/metabolismo , Putrescina/farmacologia , Espermidina/farmacologia , Espermidina Sintase/antagonistas & inibidores , Espermidina Sintase/metabolismo , Espermina/metabolismo , Espermina/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/metabolismoRESUMO
alpha-Difluoromethylornithine (DFMO), the specific and irreversible inhibitor of ornithine decarboxylase (ODC), was able to induce the arrest of proliferation in Leishmania mexicana and ODC-transformed Trypanosoma cruzi cultures grown in a semi-defined medium essentially free of polyamines. Conversely, Crithidia fasciculata and Phytomonas 274 were not affected by the inhibitor. The drug-resistance of Crithidia and Phytomonas was neither caused by an impairment of DFMO uptake nor by a decrease of the enzyme affinity for the inhibitor. We were also able to rule out the possibility of ODC overexpression in the drug-tolerant parasites. The measurements of ODC metabolic turnover indicated that the enzymes from Crithidia and Phytomonas have a short half-life of 20-40 min, while ODC from Leishmania and transgenic Trypanosoma cruzi are rather stable with a half-life longer than 6 hours. Analyses of polyamine internal pools under different growth conditions have shown that DFMO was able to markedly decrease the levels of putrescine and spermidine in all parasites, but the depletion of spermidine was higher in trypanosomatids containing an ODC with slow turnover. Our results suggest that in these parasites cultivated in the presence of the drug, spermidine might decrease below critical levels needed to maintain trypanothione concentrations or other conditions essential for normal proliferation.
Assuntos
Eflornitina/farmacologia , Ornitina Descarboxilase/genética , Ornitina Descarboxilase/metabolismo , Trypanosomatina/efeitos dos fármacos , Trypanosomatina/enzimologia , Animais , Crithidia fasciculata/efeitos dos fármacos , Crithidia fasciculata/enzimologia , Crithidia fasciculata/crescimento & desenvolvimento , Cicloeximida/farmacologia , Cinética , Leishmania mexicana/efeitos dos fármacos , Leishmania mexicana/enzimologia , Leishmania mexicana/crescimento & desenvolvimento , Putrescina/metabolismo , Espermidina/metabolismo , Fatores de Tempo , Trypanosoma cruzi/efeitos dos fármacos , Trypanosoma cruzi/enzimologia , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosomatina/crescimento & desenvolvimentoRESUMO
Trypanosoma cruzi, a pathogenic protozoan causing Chagas disease, lacks ornithine decarboxylase (ODC), the enzyme catalyzing the first step of polyamine biosynthetic pathway in eukaryotic cells. Our results indicate that the auxotrophy for diamines of T. cruzi epimastigotes is due to the absence of an active ODC gene in these parasites and not to the inability for the expression of this gene. The introduction of an exogenous complete coding region from Crithidia fasciculata ODC gene inserted in an expression vector specific for trypanosomatids induces the normal expression of the foreign genetic information allowing the transformed T. cruzi to overcome the exogenous polyamine requirement for growth. The enzyme expressed in the transformed parasites has shown a considerably extended metabolic stability. The loss of ODC activity in T. cruzi might be related to the parasite adaptation to the intracellular stages of its life cycle.
Assuntos
Regulação Enzimológica da Expressão Gênica , Ornitina Descarboxilase/genética , Trypanosoma cruzi/genética , Animais , Genes de Protozoários , Ornitina Descarboxilase/biossíntese , Trypanosoma cruzi/metabolismoRESUMO
Proliferation of Leishmania mexicana promastigotes in synthetic medium can be blocked by the depletion of intracellular polyamine pools induced by the presence of D,L-alpha-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ornithine decarboxylase (ODC). Here we report that DFMO-resistant cell lines growing normally at DFMO levels of 10 mM have been obtained from non-proliferating cultures after a single-step selection in the presence of high concentrations of the drug. The DFMO-resistant promastigotes underwent a morphological transformation into an 'amastigote-like' form after incubation for several hours at gradually increasing temperatures up to 35 degrees C. The uptake of DFMO was not significantly altered in the drug-resistant cell lines but in both cases (promastigote and 'amastigote-like' forms) the ODC specific activity was increased approx. 15-fold over the normal enzymic levels found in the wild-type Leishmania. The enzyme affinities for its substrate and for DFMO gave very similar values in the drug-resistant promastigotes and the wild-type parasites. In contrast, ODC from the 'amastigote-like' Leishmania showed a higher affinity for ornithine and a decreased capacity for the binding of DFMO. An 80-fold amplification of the ODC gene and a corresponding increase in its transcripts have been detected in both DFMO-resistant Leishmania cell lines. The drug-resistant phenotypes with their characteristic morphologies, the increased levels of ODC activity and the amplification of the ODC gene have been stable for at least 6 months in the absence of selective pressure.
Assuntos
Eflornitina/farmacologia , Inibidores Enzimáticos/farmacologia , Leishmania mexicana/efeitos dos fármacos , Animais , Resistência a Medicamentos , Amplificação de Genes , Leishmania mexicana/citologia , Leishmania mexicana/enzimologia , Ornitina Descarboxilase/genética , Inibidores da Ornitina Descarboxilase , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
The catalytic properties of ornithine decarboxylase (ODC) from Leishmania mexicana as well as the interaction with its cofactor pyridoxal 5'-phosphate (PLP) and the irreversible inhibitor alpha-difluoromethylornithine (DFMO) have been studied using partially purified preparations of the enzyme obtained from parasite promastigotes. Leishmania extracts prepared in the presence of saturating concentrations of PLP yielded an enzyme considerably more resistant to heat inactivation and with a three-fold higher activity than the ODC obtained without the addition of cofactor. The complete removal of PLP by treatment with hydroxylamine yielded the apoenzyme which shows an absolute requirement for PLP to recover its enzymatic activity. The Km values for L-ornithine and PLP were 0.7 mM and 25 microM, respectively, while Ki for DFMO was 0.2 mM. The restoration of ODC activity from apoenzyme and cofactor seems to involve time and temperature-dependent activation processes. L. mexicana ODC has an apparent molecular mass of 240 +/- 20 kDa.
Assuntos
Eflornitina/farmacologia , Leishmania mexicana/enzimologia , Ornitina Descarboxilase/metabolismo , Fosfato de Piridoxal/farmacologia , Animais , Apoenzimas/metabolismo , Catálise , Cromatografia em Gel , Ativação Enzimática/efeitos dos fármacos , Reativadores Enzimáticos/farmacologia , Temperatura Alta , Hidroxilamina , Hidroxilaminas/farmacologia , Peso Molecular , Ornitina Descarboxilase/química , Inibidores da Ornitina DescarboxilaseRESUMO
Putrescine uptake of Leishmania mexicana promastigotes is tightly regulated by polyamine intracellular concentrations. This uptake, markedly stimulated after parasite treatment with alpha-difluoromethylornithine (DFMO) for 48 to 72 hrs., was strongly repressed by exposure of Leishmania cultures to exogenous putrescine or its derivative 1,4-dimethylputrescine. In contrast, spermidine, spermine, diaminopropane and cadaverine were unable to decrease putrescine transport. Both, the uptake induction as well as its specific feedback repression by increased levels of endogenous putrescine requires protein synthesis since they were abolished after addition of cycloheximide for several hours. Our results seem to indicate that putrescine transporter is a stable and specific protein which can be reversibly inactivated by a relatively unstable repressor.
Assuntos
Leishmania mexicana/metabolismo , Putrescina/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Cicloeximida/farmacologia , Eflornitina/farmacologia , Retroalimentação , Cinética , Leishmania mexicana/efeitos dos fármacos , Leishmania mexicana/crescimento & desenvolvimento , Poliaminas/metabolismo , Poliaminas/farmacologiaRESUMO
Putrescine uptake in Trypanosoma cruzi epimastigotes is 10 to 50-fold higher than in Leishmania mexicana or Crithidia fasciculata. Polyamine transport in all these trypanosomatids is an energy-dependent process strongly inhibited by the presence of 2,4-dinitrophenol or KCN. Putrescine uptake in T. cruzi and L. mexicana was markedly decreased by the proton ionophore carbonylcyanide m-chlorophenylhydrazone but it was not affected by ouabain, a Na(+)-K+ pump inhibitor. The depletion of intracellular polyamines by treatment of parasite cultures with alpha-difluoromethylornithine elicited a marked induction of putrescine uptake in L. mexicana and C. fasciculata by increasing considerably the Vmax of this process. Conversely, the uptake of putrescine in T. cruzi was essentially unchanged by the same treatment. The differential regulation of putrescine transport in T. cruzi might be related to some distinctive features of polyamine metabolism in this parasite.
Assuntos
Crithidia fasciculata/metabolismo , Leishmania mexicana/metabolismo , Putrescina/metabolismo , Trypanosoma cruzi/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Crithidia fasciculata/efeitos dos fármacos , Eflornitina/farmacologia , Homeostase , Cinética , Leishmania mexicana/efeitos dos fármacos , Temperatura , Trypanosoma cruzi/efeitos dos fármacosRESUMO
Ornithine decarboxylase (ODC) of Crithidia fasciculata extracts shows maximal activity during exponential growth of the parasite and decreases markedly in the stationary phase. The inhibition of protein synthesis by cycloheximide evoked a rapid loss of enzyme activity with a half-life of about 30 min. Upon removal of DFMO from Crithidia cultures treated with the drug for 24 h, the ODC activity increased at the same rate as total protein synthesis. The addition of putrescine at high concentrations to parasites cultivated in a synthetic medium showed that Crithidia ODC levels were not reduced by polyamines.
Assuntos
Crithidia fasciculata/enzimologia , Regulação para Baixo , Ornitina Descarboxilase/metabolismo , Poliaminas/metabolismo , Animais , Catálise , Crithidia fasciculata/crescimento & desenvolvimento , Putrescina/metabolismoRESUMO
Repeated treatments of Leishmania mexicana promastigote cultures with a-difluoromethylornithine could not block proliferation when the parasite was grown in a rich medium. Although the irreversible inhibitor of ornithine decarboxylase was able to abolish the enzymatic activity under these conditions, polyamine depletion was only partial probably due to the uptake of these substances from the external medium. Conversely, when Leishmania was cultivated in a defined medium essentially free of polyamines, a-difluoromethylornithine was able to decrease the growth rate and proliferation was arrested after several passages in the presence of the drug. Parasite multiplication could be resumed by addition of exogenous polyamines, and a strict correlation between Leishmania promastigote growth and intracellular levels of spermidine was observed.
Assuntos
Eflornitina/farmacologia , Leishmania mexicana/fisiologia , Poliaminas/metabolismo , Animais , Cinética , Leishmania mexicana/efeitos dos fármacos , Ornitina Descarboxilase/metabolismo , Poliaminas/farmacologia , Reprodução/efeitos dos fármacosRESUMO
Nifurtimox (NF) and benznidazole (BZ), drugs used in the treatment of Chagas' disease, did not inhibit protein biosynthesis in in vitro homologous cell-free systems isolated from Trypanosoma cruzi and Crithidia fasciculata; nevertheless, their addition to growing cultures caused polyribosomal depolymerization. On the other hand, Berenil, Antrycide and suramin, used against African trypanosomiasis, inhibited protein biosynthesis in vitro but did not affect ribosomal distribution, probably due to low permeability to the drugs. The results suggest that the inhibition by NF and BZ of protein synthesis, measured as [14C]leucine incorporation by other authors, is indirect, probably through inhibition of nucleic acid synthesis and energy metabolism.