Polyamine metabolism in Trypanosoma cruzi: studies on the expression and regulation of heterologous genes involved in polyamine biosynthesis.

Biochemical studies have shown that Trypanosoma cruzi and Toxoplasma gondii are the only eukaryotic organisms so far described which are auxotrophic for polyamines. Both parasites are unable to carry out the de novo biosynthesis of putrescine, and therefore they need the addition of exogenous polyamines to the culture medium for their normal proliferation. Further investigations at the molecular level have demonstrated that the wild-type T. cruzi genome does not contain ornithine or arginine decarboxylase-like nucleic acid sequences, and that the corresponding genes have been presumably lost during evolution. Since T. cruzi behaves as a deletion mutant for ornithine decarboxylase (ODC) and arginine decarboxylase (ADC) genes, this parasite has been selected to study the regulation of the expression of heterologous genes involved in polyamine biosynthesis in other organisms. The resulting transgenic parasites have been useful tools to analyze the different stages of gene expression after transformation, as well as the mechanisms of drug resistance induction and the post-translational processing of enzyme precursors.

Spermidine is essential for normal proliferation of trypanosomatid protozoa.

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.

Sensitivity of trypanosomatid protozoa to DFMO and metabolic turnover of ornithine decarboxylase.

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.

Trypanosoma cruzi epimastigotes lack ornithine decarboxylase but can express a foreign gene encoding this enzyme.

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.

Characterization of a novel translational inhibitor from Leishmania mexicana promastigotes.

An inhibitory activity blocking protein synthesis elongation in several eukaryotic systems has been detected in Leishmania mexicana extracts. This factor, which competes with aminoacylation of tRNA and also affects the subsequent polymerization step, is a strong inhibitor of polypeptide synthesis induced by poly U in wheat-germ extracts or by endogenous mRNAs in rat liver cell-free systems. The purified translational inhibitor has shown to be essentially free of proteins. Several chemical and biochemical properties of the inhibition factor have supported the conclusion that it behaves as a 200 bases RNA with a high content of secondary structure.

Streptomycin bactericidal action is dependent on polyamine endogenous levels in E. coli.

E. coli polyamine-supplemented and depleted cultures showed an important difference in survival to streptomycin; the bactericidal effect of the antibiotic was remarkably higher in cells with normal levels of polyamines. Similar results were observed with kanamycin. Analysis of the polyamine-containing cells pulse-labelled with 35S-methionine during streptomycin action indicated that the amounts of newly-synthesized peptides in various subcellular fractions was different from the amounts formed in the untreated controls; the most dramatic change was found in the residual particulate fraction where the antibiotic treatment caused a 3-fold increase of radioactive proteins. On the contrary, equivalent amounts of labelled peptides were detected in the different fractions prepared from polyamine-depleted bacteria incubated with or without antibiotic. In this case the corresponding residual fraction was only slightly increased. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the different fractions showed some changes elicited by streptomycin in the protein patterns of polyamine-containing bacteria, especially in the residual fractions. The electrophoretic profile corresponding to deprived cells was very similar in all cases. The role of polyamines in the conformation of the outer membrane and in the correct assembly of ribosomes is discussed on account of the enhancing effect of these polycations on the bactericidal action of streptomycin.

Alpha-difluoromethylornithine-resistant cell lines obtained after one-step selection of Leishmania mexicana promastigote cultures.

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.

Cloning of a trypanosomatid gene coding for an ornithine decarboxylase that is metabolically unstable even though it lacks the C-terminal degradation domain.

Mammalian ornithine decarboxylase (ODC) is among the most labile of cellular proteins, with a half-life of usually less than an hour. Like other short-lived proteins ODC is degraded by the 26S proteasome. Its degradation is not triggered by ubiquitination, but is stimulated by the binding of an inducible protein, antizyme. Truncations and mutations in the C terminus of mammalian ODC have been shown to prevent the rapid turnover of the enzyme, demonstrating the presence of a degradation signal in this region. Moreover, ODCs from the trypanosomatid parasites Trypanosoma brucei and Leishmania donovani, which lack this C-terminal domain, are metabolically stable, and recombination of T. brucei ODC with the C terminus of mammalian ODC confers a short half-life to the fusion protein when expressed in mammalian cells. In the present study we have cloned and sequenced the ODC gene from the trypanosomatid Crithidia fasciculata. To our knowledge, this is the first protozoan shown to have an ODC with a rapid turnover. The sequence analysis revealed a high homology between C. fasciculata ODC and L. donovani ODC, despite the difference in stability. We demonstrate that C. fasciculata ODC has a very rapid turnover even when expressed in mammalian cells. Moreover, ODC from C. fasciculata is shown to lack the C-terminal degradation domain of mammalian ODC. Our findings indicate that C. fasciculata ODC contains unique signals, targeting the enzyme for rapid degradation not only in the parasite but also in mammalian cells.

Effect of polyamines on plasmid-mediated kanamycin resistance and kanamycin phosphotransferase gene expression in Escherichia coli.

The emergence of kanamycin resistance in a polyamine-deficient mutant of E. coli transformed with a plasmid encoding the kanamycin phosphotransferase gene has been studied. The initial inhibition of growth and protein synthesis caused by the addition of the antibiotic could be reversed earlier in polyamine-supplemented bacteria than in those depleted of the organic bases. Concomitantly, we have observed that the increase of kanamycin phosphotransferase activity evoking the antibiotic resistance was higher in bacteria cultivated in the presence of putrescine. This result seems to depend exclusively on the enhanced capacity of the translation process in bacteria grown with polyamines since the transcription of phosphotransferase gene was higher in cells subjected to polyamine starvation.

Ornithine decarboxylase from Leishmania mexicana promastigotes: interaction with pyridoxal 5'-phosphate and alpha-difluoromethylornithine.

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.

Regulation of putrescine uptake in Leishmania mexicana promastigotes.

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.

Inhibition of in vivo transcription by actinomycin D treatment of ethylenediaminetetraacetic acid-permeabilized Escherichia coli: effects on bacteriophage proliferation.

Rifampicin, which is able to block DNA-dependent RNA synthesis, has been widely used to selectively inhibit host protein synthesis in RNA bacteriophage-infected Escherichia coli without affecting the viral specific protein synthesis. However, in many cases it is necessary to increase rifampicin levels to 200 micrograms/ml in order to obtain an almost complete suppression of bacterial protein synthesis, and these high antibiotic concentrations cause at the same time a strong inhibition of phage proliferation resulting in a 50- to 100-fold reduction of phage yields. We have partially avoided this difficulty by using actinomycin D after permeabilization of bacteria by a brief incubation with EDTA. To optimize the method the effects of changing EDTA and actinomycin concentrations as well as the duration of the permeabilization period have been studied. With this procedure it has been possible to shut off bacterial RNA and protein synthesis with phage yields about 10 times higher than those observed in the presence of high levels of rifampicin. The usefulness of the described method is particularly evident when working with rifampicin-resistant strains of E. coli.

Polyamines prevent DFMO-mediated inhibition of angiogenesis.

Tumor growth mainly depend on formation of new blood vessels. DFMO (alpha-difluoromethylornithine), an inhibitor of polyamine biosynthesis, inhibits tumor growth in many animal tumors. Our investigation was to evaluate the requirement of polyamines for induction of angiogenesis by tumor cells and spleen lymphocytes from tumor-bearing mice. In this regard, we have added DFMO to cell cultures. The neovascular response induced either by tumor cells or spleen lymphocytes was completely abrogated. This inhibition could be reversed by the addition of exogenous putrescine. These findings suggest that the effect of DFMO on angiogenesis is, in part, mediated by the inhibition of polyamine biosynthesis.

Polyamines modulate streptomycin-induced mistranslation in Escherichia coli.

The effects of intracellular levels of polyamines on both the in vivo inhibition of protein synthesis and the decrease of translation accuracy induced by streptomycin have been studied in polyamine-auxotrophic strains of Escherichia coli infected with the MS2 bacteriophage. The amount of viral coat protein formed was strongly reduced upon addition of increasing concentrations of streptomycin to polyamine-supplemented bacteria. In contrast, the antibiotic almost did not inhibit coat protein synthesis in polyamine-starved cells. The increase of mistranslation frequency elicited by streptomycin was only observed in bacteria grown with putrescine. In these cells several coat protein-satellites were detected after two-dimensional gel electrophoresis. These proteins, more basic than the normal MS2 coat protein, contain multiple substitutions of lysine for asparagine.

Differential regulation of putrescine uptake in Trypanosoma cruzi and other trypanosomatids.

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.

Ornithine decarboxylase from Crithidia fasciculata is metabolically unstable and resistant to polyamine down-regulation.

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.

Control of Leishmania mexicana proliferation by modulation of polyamine intracellular levels.

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.

Stable ornithine decarboxylase in promastigotes of Leishmania mexicana mexicana.

Studies on the decarboxylation of ornithine in Leishmania mexicana have shown that this activity corresponds to a true ornithine decarboxylase rather than to an oxidative decarboxylation or aminotransferase reaction, both of which also give rise to the release of CO2. The stoichiometric relationship between substrate and products has indicated that extracts of L. mexicana were able to catalyse the formation of an unknown compound besides putrescine and CO2. The addition of cycloheximide to cultures of L. mexicana allowed us to demonstrate that ornithine decarboxylase degradation in vivo was extremely slow in this parasite. This remarkable stability of the enzyme is only comparable to that found in Trypanosoma brucei and contrasts with the high turnover rate of ornithine decarboxylases of different mammalian cells.

Polyamines and regulation of ornithine biosynthesis in Escherichia coli.

The growth rate of several polyamine-deficient mutants of Escherichia coli was very low in minimal medium and increased markedly upon the addition of putrescine, spermidine, arginine, citrulline, or argininosuccinic acid. The endogenous content of polyamines was not significantly altered by the supplementation of polyamine-starved cultures with arginine or its precursors. In contrast, these compounds as well as putrescine or spermidine caused a 40-fold reduction in intracellular ornithine levels when added to polyamine-depleted bacteria. In vivo experiments with radioactive glutamic acid as a precursor and in vitro assays of the related enzymes showed that the decrease in ornithine levels was due to the inhibition of its biosynthesis rather than to an increase in its conversion to citrulline or delta 1-pyrroline-5-carboxylic acid and proline. High endogenous concentrations of ornithine were toxic for the E. coli strains tested. The described results indicate that the stimulatory effect of putrescine and spermidine on the growth of certain polyamine-starved bacteria may be partially due to the control of ornithine biosynthesis by polyamines.

Inhibition of protein synthesis by aminoglycoside antibiotics in polyamine-requiring bacteria.

The effect of streptomycin and other aminoglycosides on protein synthesis has been studied using various streptomycin-sensitive strains unable to synthesize polyamines. We have confirmed and extended our previous results showing that the strong inhibition of translation caused by the antibiotic in polyamine-supplemented bacteria was markedly reduced in polyamine-starved cells. The analysis of polypeptides synthesized in the absence and presence of streptomycin in bacteria grown with and without putrescine has shown that the antibiotic provoked the accumulation of low molecular weight peptides partially bound to ribosomes in polyamine-unstarved cells. On the contrary, the drug did not induce major alterations in the patterns of proteins obtained from polyamine-depleted bacteria. The addition of the antibiotic did not evoke any change of proteolytic activity.