Presence and properties of thymidylate synthase in trypanosomatids.

High speed centrifugal supernatant fractions of homogenates of a number of trypanosomatids were assayed for thymidylate synthase (5,10-methylene-tetrahydrofolate: dUMP C-methyltransferase, EC 2.1.1.45) activity using the method of Lomax and Greenberg (1967) J. Biol. Chem. 242, 109-113). Similar activities were detected in Crithidia fasciculata, Crithidia oncopelti, the blood forms of Trypanosoma brucei, Trypansoma congolense and Trypanosoma lewisi and the blood, intracellular and culture forms of Trypanosoma cruzi, suggesting that all species synthesize at least some thymidylate de novo. The properties of the activities in C. fasciculata and the three forms of T. cruzi were compared with those of the isofunctional bacterial and mammalian enzymes. The trypanosotamid enzyme was inhibited by Mg2+, was much more sensitive to mercaptoethanol, had higher apparent Km values for substrate (dUMP) and cofactor (tetrahydrofolate), had a higher apparent molecular weight and was markedly more sensitive to inhibition by suramin. It is, therefore a possible target for chemotherapeutic attack, either on its own or in combination with a dihydrofolate reductase inhibitor. No evidence was obtained for the regulation of the trypanosomatid enzyme, either by its product, dTMP, or by dTDP or dTTp. This result agrees with previous studies which suggested that in trypanosomatids, the level of dTMP was regulated, at least in part, by a catabolic pathway consisting of a thymidylate phosphatase and a thymidine phosphorylase which degraded the excess of dTMP to thymine.     

Characteristics of cytidine aminohydrolase activity in Trypanosoma cruzi and Crithidia fasciculata

Cytidine deaminase (cytidine aminohydrolase, 3.5.4.5) is present in Crithidia fasciculata (a mosquito parasite) and in Trypanosoma cruzi (a human pathogen). The enzyme from C. fasciculata deaminated both cytidine and deoxycytidine, the affinity for the former being much lower than the latter. Affinities for both substrates are equal for the T. cruzi enzyme. The production of the enzyme in C. fasciculata was significantly stimulated by the addition of a number of pyrimidine nucleosides (cytidine, uridine, 5-bromouridine, thymidine, orotidine) to the culture media. Only cytidine stimulated enzyme production in T. cruzi. The enzyme from both organisms was unstable in air, even in the frozen state. Stabilization was achieved under anaerobic conditions.     

Thymine and Thymidine Uptake by Haemophilus influenzae and the Labeling of Deoxyribonucleic Acid

The influence of ribo- and deoxyribonucleosides and ribo- and deoxyribonucleotides on the uptake of radiolabeled thymidine and thymine by Haemophilus influenzae during growth was investigated. A nucleoside-degrading enzyme similar to that reported in Escherichia coli was found to break down thymidine unless other nucleosides were present to divert its action. The presence of other nucleosides permitted a nearly quantitative uptake of even low levels of thymidine. This quantitative uptake of thymidine in the presence of an excess of other nucleosides suggests that the uptake mechanism for thymidine is specific in this organism. Under optimal conditions, as much as 50% of the deoxyribonucleic acid (DNA) thymine was derived from exogenous thymidine. Thymine was not taken up by H. influenzae but, in the presence of purine deoxynucleosides, labeled thymine entered the cells, presumably as thymidine. Ribonucleosides or ribonucleotides inhibited thymine conversion to thymidine, but, as noted above, they were degraded by a cellular enzyme. Thus, unless the ribonucleoside level was excessively high, a cell level of growth was reached at which the inhibiting ribonucleoside was broken down and labeled thymine appeared in the cells at an increasing rate. Twenty-five per cent of the DNA thymine of this organism was labeled with exogenous thymine. The information noted above serves as the basis for isotopically labeling the DNA.     

Alternative metabolic fates of thymine nucleotides in human cells.

Three types of experiments have been used to study the metabolism of thymine nucleotides by human cells. (1) Cells were labelled continuously with [3H]thymidine and the incorporation of label into DNA compared with the specific radioactivities of pools of individual thymine nucleotides separated by chromatography on polyethylene-imine-cellulose. (2) Cellular thymine nucleotides were labelled with [3H]thymidine at 13 degrees C, followed by incubation at 37 degrees C in unlabelled medium. Incorporation of label into DNA and loss of label from the nucleotide pools were monitored during the 'chase' period at 37 degrees C. (3) The experiments described in (2) above were repeated in the presence of the DNA-synthesis inhibitor cytosine arabinoside, in order to demonstrate more clearly and to quantify degradative pathways for thymine nucleotides. In phytohaemagglutinin-stimulated lymphocytes and in bone-marrow cells, only a proportion (25-60%) of labelled thymine nucleotide was incorporated into DNA, the rest being rapidly degraded and lost from the cell. In contrast, an established cell line (HPB-ALL) from a patient with acute lymphoblastic leukaemia of thymic origin incorporated 100% of its exogenously labelled thymine nucleotides into DNA. These results indicated that alternative metabolic routes are open to thymine nucleotides in human cells. In lymphocytes from patients with megaloblastic anaemia and in normal lymphocytes treated with methotrexate, the utilization of labelled thymine nucleotides for DNA synthesis was more efficient than in controls. These results offer an explanation for the observation of a normal pool of thymidine triphosphate in the cells of patients with untreated megaloblastic anaemia even though the amount of this compound available for DNA synthesis appears to be decreased.     

Pyrimidine metabolism in Acinetobacter calcoaceticus

The metabolism of thymine, thymidine, uracil, and uridine has been investigated in five different strains of Acinetobacter calcoaceticus. Attempts to isolate thymine and thymidine auxotrophic mutants were not successful. Consistent with this finding was the observation that uptake of radioactive thymine or thymidine could not be demonstrated. Search for enzymes capable of transforming thymine via thymidine to thymidine-5'-monophosphate in crude extracts was performed, and the following enzymes were absent judging from enzyme assays: thymidine phosphorylase (EC 2.4.2.4), trans-N-deoxyribosylase (EC 2.4.2.6), and thymidine kinase (EC 2.7.1.21). The enzymes responsible for the phosphorylation of thymidine-5'-monophosphate to thymidine-5'-triphosphate were present in crude extracts. Radioactive uracil was readily incorporated into both ribonucleic acid and deoxyribonucleic acid, the ratio being 6:1, and radioactivity was found only in pyrimidine bases. No uptake of uridine could be demonstrated. Uridine-5'-monophosphate pyrophosphorylase (EC 2.4.2.9) activity was detected in crude extracts, suggesting that uracil is converted directly to uridine-5'-monophosphate which is then phosphorylated to uridine-5'-triphosphate or transformed to other ribo- and deoxypyrimidine nucleotides.     

On the production of glycerol and l-alanine during the aerobic fermentation of glucose by trypanosomatids

Abstract Glucose consumption and catabolite production by thick suspensions of Trypanosoma cruzi, Leishmania mexicana and Crithidia fasciculata were similar under aerobic and anaerobic conditions, indicating lack of Pasteur effect. Succinate was the main product for L. mexicana and C. fasciculata ; the latter also produced similar amounts of ethanol. T. cruzi produced succinate and l -alanine to a similar extent. l -Alanine was also a major product of L. mexicana , but was neither produced, nor consumed, by C. fasciculata . Small amounts of glycerol were produced by L. mexicana and C. fasciculata , but not by T. cruzi , which had no detectable NAD-dependent sn -glycerol-3-phosphate dehydrogenase activity.     

Trypanothione S-transferase activity in a trypanosomatid ribosomal elongation factor 1B

Trypanothione is a thiol unique to the Kinetoplastida and has been shown to be a vital component of their antioxidant defenses. However, little is known as to the role of trypanothione in xenobiotic metabolism. A trypanothione S-transferase activity was detected in extracts of Leishmania major, L. infantum, L. tarentolae, Trypanosoma brucei, and Crithidia fasciculata, but not Trypanosoma cruzi. No glutathione S-transferase activity was detected in any of these parasites. Trypanothione S-transferase was purified from C. fasciculata and shown to be a hexadecameric complex of three subunits with a relative molecular weight of 650,000. This enzyme complex was specific for the thiols trypanothione and glutathionylspermidine and only used 1-chloro-2,4-dinitrobenzene from a range of glutathione S-transferase substrates. Peptide sequencing revealed that the three components were the alpha, beta, and gamma subunits of ribosomal eukaryotic elongation factor 1B (eEF1B). Partial dissociation of the complex suggested that the S-transferase activity was associated with the gamma subunit. Moreover, Cibacron blue was found to be a tight binding inhibitor and reactive blue 4 an irreversible time-dependent inhibitor that covalently modified only the gamma subunit. The rate of inactivation by reactive blue 4 was increased more than 600-fold in the presence of trypanothione, and Cibacron blue protected the enzyme from inactivation by 1-chloro-2,4-dinitrobenzene, confirming that these dyes interact with the active site region. Two eEF1Bgamma genes were cloned from C. fasciculata, but recombinant C. fasciculata eEF1Bgamma had no S-transferase activity, suggesting that eEF1Bgamma is unstable in the absence of the other subunits.     

Trypanosoma cruzi oleate desaturase: molecular characterization and comparative analysis in other trypanosomatids

Trypanosoma cruzi lipids contain a high content of unsaturated fatty acids, primarily oleic acid (C18:1) and linoleic acid (C18:2). Previous data suggest that this parasite is able to convert oleic acid into linoleic acid; humans are not able to do this. Presently, we show that T. cruzi has a gene with high similarity to the delta12 (omega6)-oleate desaturase from plants. Northern blot analysis of the oleate desaturase gene from T. cruzi (OD(Tc)) indicated that this gene is transcribed in epimastigote, amastigote, and trypomastigote forms. Pulsed-field analysis showed that OD(Tc) is located at distinct chromosomal bands on distinct T. cruzi phylogenetic groups. In addition, the chromoblot analysis demonstrated the presence of homologous OD(Tc) genes in several trypanosomatids; namely, Crithidia fasciculata, Herpetomonas megaseliae, Leptomonas seymouri, Trypanosoma freitasi, Trypanosoma rangeli, Trypanosoma lewisi, Blastocrithidia sp., Leishmania amazonensis, Endotrypanum schaudinni, and Trypanosoma conorhini. The native OD(Tc) activity was detected by metabolic labeling and analysis of total fatty acids from epimastigotes and trypomastigotes of T. cruzi, coanomastigotes of C. fasciculata, and promastigotes of L. amazonensis, H. megaseliae, and L. seymouri. The fact that the enzyme oleate desaturase is not present in humans makes it an ideal molecular target for the development of new chemotherapeutic approaches against Chagas disease.     

Pyrimidine deoxyribonucleotide metabolism in Acholeplasma laidlawii B-PG9.

Extracts of Acholeplasma laidlawii B-PG9 were examined for the enzymes associated with the interconversion of the pyrimidine deoxyribonucleotides and the biosynthesis of thymidine nucleotides. A. laidlawii B-PG9 possessed deaminases for deoxycytidine and dCMP, pyrophosphatases for dUTP, phosphorylases for thymidine and uridine, and a membrane-associated pyrimidine deoxyribonucleoside monophosphate phosphatase activity. The role these enzyme activities have in the generation of deoxyribose-1-phosphate during growth may explain the ability of A. laidlawii B-PG9 to utilize either thymine or thymidine for biosynthesis.     

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.     

THYMIDINE TRIPHOSPHATE SYNTHESIS IN TETRAHYMENA : I. Studies on Thymidine Kinase

The amount of thymidine-H³ converted to thymidine-H³ monophosphate in 30 min formed the basis for assays of thymidine kinase in cell extracts from Tetrahymena pyriformis. The optimal concentration of adenosine triphosphate is lower than that required by other cell types. Thymidine triphosphate does not exercise any feedback control of the enzyme. Other deoxyprimidine nucleotides were tested, but these also failed to exhibit any feedback inhibition. At suboptimal adenosine triphosphate levels, thymidine triphosphate and other deoxypyrimidine nucleotides stimulate the reaction, suggesting that these nucleotides may act either directly or indirectly as phosphate donors in the crude enzyme preparations. This possibility was affirmed when thymidine triphosphate and deoxycytidine triphosphate were shown to be capable of limited phosphorylation of thymidine. Comparison of enzymatic activities in logarithmically growing culture and stationary phase culture, in which nuclear DNA synthesis has virtually ceased, reveals no change in enzymatic activity. The results suggest that thymidine kinase is a constitutive enzyme in Tetrahymena.     

Thymidine and Thymine Incorporation into Deoxyribonucleic Acid: Inhibition and Repression by Uridine of Thymidine Phosphorylase of Escherichia coli

Thymidine is poorly incorporated into deoxyribonucleic acid (DNA) of Escherichia coli. Its incorporation is greatly increased by uridine, which acts in two ways. Primarily, uridine competitively inhibits thymidine phosphorylase (E.C.2.4.4), and thereby prevents the degradation of thymidine to thymine which is not incorporated into normally growing E. coli. Uridine also inhibits induction of the enzyme by thymidine. It prevents the actual inducer, probably a deoxyribose phosphate, from being formed rather than competing for a site on the repressor. The inhibition of thymidine phosphorylase by uridine also accounts for inhibition by uracil compounds of thymine incorporation into thymine-requiring mutants. Deoxyadenosine also increases the incorporation of thymidine, by competitively inhibiting thymidine phosphorylase. Deoxyadenosine induces the enzyme, in contrast to uridine. But this is offset by a transfer of deoxyribose from deoxyadenosine to thymine. Thus, deoxyadenosine permits incorporation of thymine into DNA, even in cells induced for thymidine phosphorylase. This incorporation of thymine in the presence of deoxyadenosine did not occur in a thymidine phosphorylase-negative mutant; thus, the utilization of thymine seems to proceed by way of thymidine phosphorylase, followed by thymidine kinase. These results are consistent with the data of others in suggesting that wild-type E. coli cells fail to utilize thymine because they lack a pool of deoxyribose phosphates, the latter being necessary for conversion of thymine to thymidine by thymidine phosphorylase.     

Alteration of the cell surface acid phosphatase concomitant with the morphological transformation in Trypanosoma cruzi

Acid phosphatase activity in Trypanosoma cruzi was found to be located on the external surface of the plasma membranes. Both specific activity and activity per cell of this bound enzyme were significantly higher in the cells of amastigote (an intracellular form) than that of trypomastigote (a bloodstream form) and epimastigote (culture form). During the transformation of epimastigotes to amastigotes in vitro the activity of surface acid phosphatase was elevated concomitant with the increase in population of amastigotes. These results were interpreted as that the elevated enzyme activity is required for the intracellular parasitization of this organism or is a consequence of the morphological transformation.     

Disruption of the Crithidia fasciculata RNH1 gene results in the loss of two active forms of ribonuclease H.

Both prokaryotic and eukaryotic cells contain multiple forms of ribonuclease H, a ribonuclease that specifically degrades the RNA strand of RNA-DNA hybrids and which has been implicated in the processing of initiator RNAs and in the removal of RNA primers from Okazaki fragments. The Crithidia fasciculata RNH1 gene encodes an RNase H and was shown to be a single-copy gene in this diploid trypanosomatid. The RNH1 gene has been disrupted by targeted gene disruption using hygromycin or G418 drug-resistance cassettes. Major active forms of RNase H (38 and 45 kDa) were observed on activity gels of extracts of wild-type cells or cells in which one allele of RNH1 was disrupted. Both the 38 and 45 kDa activities were absent in extracts of cells in which both alleles of RNH1 were disrupted indicating that both forms of the C.fasciculata RNase H are encoded by the RNH1 gene.     

Uptake and metabolism of nucleosides by embryos of the sea urchin Strongylocentrotus purpuratus

Uptake and metabolism of thymidine and adenosine have been studied in embryos of the sea urchin Strongylocentrotus purpuratus. Uptake of these nucleosides is found to be mutually competitive, with the Km for uptake of thymidine similar to its Ki for inhibition of adenosine uptake and vice versa. The metabolic studies show that adenosine is rapidly and completely phosphorylated upon entry, even at high exogenous concentrations which saturate the uptake mechanism. In contrast, at concentrations which saturate nucleoside uptake, thymidine becomes appreciably catabolized (up to 60%) to thymine and beta-amino-isobutyric acid in addition to its phosphorylation to thymine nucleotides. Negligible amounts of endogenous thymidine appear to remain unmetabolized following uptake in these embryos. The data provide strong in vivo evidence for separate metabolic pathways for thymidine and adenosine which have not previously been described in this organism. The observation of mutual competition during uptake, together with different routes of metabolism for these nucleosides, would suggest that the rate-limiting step in the uptake process is transport rather than metabolism. The specificity of this transport system for its nucleoside substrate has been examined in some detail in the present report. All naturally occurring nucleosides but only a limited number of nucleoside analogs are recognized by this membrane carrier. Neither purine nor pyrimidine bases are substrates for this transport system. Previous work by this laboratory has demonstrated the strict Na+-dependence of this carrier, its high affinity for nucleoside substrate, and its activation at fertilization. These observations and the substrate specificity studies of the present work together describe a unique transport system for nucleosides in sea urchin embryos which is quite different from those previously described in mammalian cells.     

Iron-containing superoxide dismutase from Crithidia fasciculata. Purification, characterization, and similarity to Leishmanial and trypanosomal enzymes

Leishmania tropica, Trypanosoma brucei, Trypanosoma cruzi, and Crithidia fasciculata have superoxide dismutases which are insensitive to cyanide and sensitive to peroxide and azide, properties characteristic of iron-containing superoxide dismutase. Studies on the superoxide dismutase of C. fasciculata have revealed that: 1) the enzyme is located in the cytosol; 2) isozymes exist; 3) the major superoxide dismutase isozyme (superoxide dismutase 2) has Mr approximately equal to 43,000 and consists of two equal-sized subunits, each of which contains 1.4 atoms of iron. Comparisons of the amino acid content of this crithidial superoxide dismutase with those of superoxide dismutases from other sources suggests that the crithidial enzyme is closely related to bacterial Fe-containing superoxide dismutases, and only distantly related to human Mn- and Cu,Zn-containing superoxide dismutases and to Euglena Fe-containing superoxide dismutase. Attempts are now underway to develop specific inhibitors of the trypanosomatid superoxide dismutase which may be of use in the treatment of leishmaniasis or trypanosomiasis.     

Thymidine phosphorylase activity and its relationship to trimethoprim in initial strains and thymidine-, thymine-dependent and trimethoprim-resistant mutants of plague microbe]

The comparative study revealed thymidine phosphorylase activity in the initial strains of a plague microbe of the field variety and in thymidine-, thymine-dependent and trimethoprim-resistant mutants of the plague microbe of other varieties. The data fully conformed to the results of the microbiological investigation of the strains' ability to grow on the nutrient media with trimethoprim in the presence of thymine and thymidine. On the basis of these results it appeared possible to divide the initial and mutant strains of the plague microbe into four arbitrary groups: initial strains of the plague microbe of all the varieties except the field ones sensitive to trimethoprim under any temperature conditions of incubation on any medium with any supplements; initial strains of the plague microbe of the field variety resistant to trimethoprim at 28 degrees C in the presence of thymine or thymidine alone; Tmpr mutants whose resistance to trimethoprim at 28 degrees C did not depend on the presence of thymine or thymidine, purine and vitamins, but depended on the presence of these substances at a temperature of 37 degrees C.     

83-kilodalton heat shock proteins of trypanosomes are potent peptide-stimulated ATPases.

A Crithidia fasciculata 83-kDa protein purified during a separate study of C. fasciculata trypanothione synthetase was shown to have ATPase activity and to belong to the hsp90 family of stress proteins. Because no ATPase activity has previously been reported for the hsp90 class, ATP utilization by C. fasciculata hsp83 was characterized: this hsp83 has an ATPase kcat of 150 min-1 and a Km of 60 microM, whereas the homologous mammalian hsp90 binds ATP but has no ATPase activity. Crithidia fasciculata hsp83 undergoes autophosphorylation on serine and threonine at a rate constant of 3.3 x 10(-3) min-1. Similar analysis was performed on recombinant Trypanosoma cruzi hsp83, and comparable ATPase parameters were obtained (kcat = 100 min-1, Km = 80 microM, kautophosphorylation = 6.3 x 10(-3) min-1). The phosphoenzyme is neither on the ATPase hydrolytic pathway nor does it affect ATPase catalytic efficiency. Both C. fasciculata and T. cruzi hsp83 show up to fivefold stimulation of ATPase activity by peptides of 6-24 amino acids.     

A 189-bp fragment of Crithidia fasciculata maxicircle DNA confers autonomous replication in Saccharomyces cerevisiae

A 189-bp fragment capable of promoting high-frequency transformation in Saccharomyces cerevisiae has been isolated from the maxicircle of the insect trypanosomatid Crithidia fasciculata. Chimeric plasmids containing this autonomously replicating sequence (ars) are maintained as extrachromosomal elements in S. cerevisiae. The nucleotide sequence of the maxicircle fragment, termed ars189, has been determined, and its position has been mapped in the maxicircle. The ars189 fragment has an A + T content of 79.4% and shows a large asymmetry in the distribution of adenine and thymine residues between the two strands. In one strand (the T strand) thymine accounts for 118 out of 189 nucleotides while adenine accounts for only 32 nucleotides. The ars189 DNA does not hybridize with minicircles, and its sequence appears to be unique in the C. fasciculata maxicircle genome. This sequence also shows extensive homology to a sequence within a 2.6-kb ars fragment of the Leishmania tarentolae maxicircle. In addition, ars189 contains two copies of a yeast consensus ars sequence (A/T)TTTATPuTTT(T/A).