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.

Protein synthesis in polyamine-deficient bacteria during amino-acid starvation.

Amino-acid starvation in polyamine-auxotrophic bacteria grown in the presence of putrescine provokes a marked inhibition of protein synthesis. This inhibition is almost completely relieved in polyamine-depleted cells. The differential behaviour of bacterial protein synthesis depending on the endogenous levels of polyamines is not due to a change in the uptake of amino acids used to measure protein synthesis, nor to the decreased growth rate of polyamine-depleted cells. During leucine starvation, cells grown with putrescine synthesized a somewhat lower amount of high-molecular-weight proteins than polyamine-depleted bacteria. In addition, cells with normal endogenous levels of polyamines accumulated significant amounts of 62 and 41 kDa polypeptides as well as several low-molecular-weight peptides.

Association of ribosomal subunits. IV. Polyamines as active components of the association factor from Bacillus stearothermophilus.

The association of ribosomal subparticles induced by several associating agents has been studied under different conditions. The following observations were made: 1. Spermidine was able to produce the association of subunits, and the concentration and temperature curves of this reaction were similar to those obtained with association factor. The product formed in the latter case was more stable. 2. The association at low Mg2+ concentration was higher with association factor than with polyamines. 3. The temperature-dependent binding of spermidine to 30-S subunits formed an active complex, which was converted into the 30S-50S couples by the addition of 50-S subparticles at low temperature. A similar behaviour has been previously shown for the complete association factor and its low molecular weight fraction. 4. The same unstable form of 30S-50S couples has been obtained either with spermidine or with the low molecular weight component (AFII) of the association factor. In both cases the protein fraction AFI was able to complete the reaction by stabilizing the subunit couple. 5. After glutaraldehyde fixation the products of the reactions with spermidine or association factor behaved in a similar way when they were submitted to long sucrose-gradient centrifugations. 6. The analysis of association factor preparations has shown that they contain spermidine as well as spermine. The polyamine levels in association factor could account for part of the total associating activity.

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.

Putrescine distribution in Escherichia coli studied in vivo by 13C nuclear magnetic resonance.

In order to study the intracellular polyamine distribution in Escherichia coli, 13C-NMR spectra of [1,4-13C]putrescine were obtained after addition of the latter to intact bacteria. The 13C-enriched methylene signal underwent line broadening. When the cells were centrifuged after 90 min the cell-bound putrescine peak had a linewidth of 23 Hz, while the supernatant liquid showed an unbound putrescine signal with a linewidth smaller than 1 Hz. By using 13C-enriched internal standards it could be shown that the linewidening was not due to the heterogeneity of the medium or to an in vivo paramagnetic effect. Cell-bound putrescine was liberated by addition of trichloroacetic acid and was therefore non-covalently linked to macromolecular cell structures. Cell-bound [13C]putrescine could be displaced by addition of an excess of [12C]putrescine. When samples of membranes, soluble protein, DNA, tRNA and ribosomes from E. coli were incubated with [1,4-13C]putrescine, strong binding was detected only in the ribosomal and membrane fractions. The ribosome-putrescine complex showed properties similar to those determined with the intact cells. By measuring the nuclear Overhauser enhancements eta, it was possible to estimate that only about 50% of the polyamine was linked to the macromolecules. Determination of the T1 values of free and ribosomal-bound putrescine allowed the calculation of a correlation time, tau c = 4 X 10(-7) s for the latter. T1 and tau c values found for the ribosome-putrescine complex were those expected for a motional regime of slowly tumbling molecules.

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.

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.

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.

A novel mechanism of resistance to alpha-difluoromethylornithine induced by cycloheximide. Growth with abnormally low levels of putrescine and spermidine.

Treatment of the chemically transformed fibroblasts BP-A31 and other cell lines with low concentrations of cycloheximide (CHM) for 72 h followed by the removal of the protein synthesis inhibitor leads to the proliferation of alpha-difluoromethylornithine (DFMO)-resistant phenotypes. These drug-resistant cells contain almost no ornithine decarboxylase (ODC) activity and concomitantly very low levels of putrescine and spermidine. Southern blot analysis and measurements of ODC activity and intracellular polyamine levels showed that the described mechanism of inducing resistance to DFMO triggered by CHM does not involve ODC gene amplification, altered transport of the drug or reduced affinity of the enzyme for DFMO.

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.