Winston E. Gutteridge

Purine and pyrimidine metabolism in the trypanosomatidae

The pathways leading to purine and pyrimidine nucleotide production in members of the family Trypanosomatidae are discussed with special emphasis on data relating to pathogenic species published from 1974 to 1983 inclusive. Trypanosomes and leishmania in general lack a de novo purine biosynthetic pathway, but have a multiplicity of possible routes for purine salvage. In contrast, pyrimidine nucleotides can be produced by either de novo or salvage pathways. The properties of these pathways in trypanosomatids are compared and contrasted with those of their hosts.

Conversion of dihydroorotate to orotate in parasitic protozoa.

The conversion of dihydroorotate to orotate, one of the key reactions in the de novo pyrimidine biosynthetic pathway, has been studied in a number of parasitic protozoa. Enzyme activities capable of carrying out this reaction were detected in six members of the Kinetoplastida (Trypanosoma brucei, Trypanosoma congolense, Trypanosoma vivax, Trypanosoma lewisi, Trypanosoma cruzi, Leishmania enriettii) and three members of the genus Plasmodium (P. knowlesi, P. berghei, P. gallinaceum). The mechanism of the reaction in the two groups of protozoa were quite distinct. In the Kinetoplastida, the enzyme is an hydroxylase which occurs in the soluble fraction of the cell and probably requires tetrahydrobiopterin for activity. In contrast, in Plasmodium, the enzyme is a dehydrogenase which is particulate, probably mitochondrial, and intimately connected to the electron transport chain to which it passes electrons directly, probably at the ubiquinone level. Neither activity is regulated by fully formed pyrimidines. The enzyme in Plasmodium is similar in mechanism to the isofunctional mammalian enzyme. However, since malarial ubiquinones are apparently different from those in the mammal and since menoctone, which is active in vivo in experimental malaria, is a good inhibitor of the malarial enzyme, it could represent a useful target for chemotherapeutic attack. The enzyme in the Kinetoplastida is quite distinct from that in the mammal so that it too apparently falls into this category, though none of the currently used antitrypanosomal drugs appears to block it activity at physiological concentrations.

A re-examination of purine and pyrimidine synthesis in the three main forms of Trypanosoma cruzi.

1. 1. Purine and pyrimidine synthesis and salvage has been re-examined in blood trypomastigote. intracellular amastigote and culture epimastigote forms of Trypanosoma cruzi. 2. 2. All forms are unable to biosynthesize the purine ring de novo and are thus dependent on salvage. Bases are preferred to nucleosides; nucleotides appear not to be salvaged. Adenine and guanine nucleotides are readily interconvertible. 3. 3. Pyrimidines can be both biosynthesized de novo and salvaged, although in vivo. only the former is likely to be of significance. Both bases and nucleosides are readily salvaged; nucleotides are not utilized to a significant extent. Pyrimidine ribonucleotides are freely interconvertible. 4. 4. Purine salvage and pyrimidine synthesis are both potential targets for chemotherapy. 5. 5. No major differences in purine and pyrimidine metabolism in the three main forms of the parasite were detected.

Purine metabolism in Trichomonas vaginalis.

The purine metabolism of several genera of parasitic protozoa, is of interest partly due to their sensitivity to purine analogues, and to differences in the enzymes of purine metabolism compared to mammalian systems [l-6]. Trichomonas vaginalis, the causative agent of trichomonal vaginitis, a mild, but very common, sexually-transmitted disease, has not been studied in this respect; there is a general lack of knowledge on the biosynthetic ability of the organism. We report here on the ability of washed-cell suspensions of T. vaginalis to salvage the purine bases, adenine and guanine, and their nucleosides, and on the absence from the organism of the ability to synthesise purines de novo and to interconvert purine nucleotides. Examination of possible purine salvage enzymes suggests that nucleoside phosphorylase and nucleoside kinase activities are responsible for the conversion of purine bases and nucleosides to nucleoside monophosphates; purine phosphoribosyltransferases are either absent or present at only very low levels.

Subcellular localization of some glycolytic enzymes in parasitic flagellated protozoa.

1. The glycolytic enzymes of Tritrichomonas foetus, unlike those of Trypanosoma brucei, are apparently not located in microbody-like organelles (glycosomes) but appear to occur in the cytosol. 2. The localization of the glycolytic enzymes in Crithidia fasciculata and in the epimastigote and trypomastigote forms of Trypanosoma cruzi is however similar to that in T. brucei. 3. No evidence was obtained for the existence of glycolytic enzyme-containing microbodies in rat liver. 4. The glycosome is probably an organelle unique to members of the Kinetoplastida.

A novel location for two enzymes of de novo pyrimidine biosynthesis in trypanosomes and Leishmania.

African trypanosomes such as Trypa~#s~~a bmcei are the causative agents for sleeping sickness in man and nagana in cattle, Trypanosoma cmzi for Chagas’ disease, and species of L&&mania for oriental sore and Kala-azar. These important protozoan parasites of man are members of the order ~netoplastida. This order is characterised by the presence of an atypical organelle rich in DNA, the kinetoplast. Another atypical organelle, the microbody-like glycosome, which contains glycolytic enzymes, has been found in T. brucei [l-3] and is probably present in all other members of the Kinetoplastida [4,5]. A common feature now emerging [S-S] is that some of the enzymes of de nova pyrimidine biosynthesis show a subcellular distribution distinct from that observed in other species (review [9]). The first 3 enzymes of this pathway (carbamoylphosphate synthetase, EC 6.3.4.16; aspartate carbamoyltransferase, EC 2.1.3.2; d~ydroorotase, EC 3.5.2.3) in the Kinetopfastida, as in other cells, are all soluble. In contrast, the fourth enzyme dihydroorotate dehydrogenase (EC 1.3.3.1) is usually particulate, mitochondrial and intimately connected to the respiratory chain; in the ~netop~astida it is soluble and is possibly a hydroxylase [lo]. The last 2 enzymes of this pathway, orotate phosphoribosyltransferase (EC 2.4.2.10, OPRTase) and orotidine-5’-phosphate decarboxylase (EC 4.1 .l. 1.23, ODCase), which are soluble in other cells, are particulate in the ~netoplastida. Here, we show that OPRTase and ODCase co-purify in isopycnic sucrose gradients with glycosomal enzymes, suggesting they are associated with microbody-like organelles, probably the glycosomes themselves.

A comparison of the interaction of anthelmintic benzimidazoles with tubulin isolated from mammalian tissue and the parasitic nematode Ascaridia galli

Colchicine and a range of anthelmintic benzimidazoles inhibited the in vitro polymerization of tubulin purified from the parasitic nematode Ascaridia galli. In most cases, this inhibition was more pronounced than that detected when these drugs were incubated with tubulin purified from mammalian tissue. In particular, oxfendazole and thiabendazole had virtually no effect on mammalian tubulin assembly whereas they were both good inhibitors of nematode tubulin polymerization. Electron microscopic examinations revealed no morphological differences between microtubules from either nematode or mammalian tissues polymerized in the presence or absence of drug, though the length and number of microtubules was reduced in the drug-incubated samples. These results show that the benzimidazole group of anthelmintics interacts specifically with nematode tubulin and that their selectivity, at least in part, is a direct consequence of such interaction.

A novel series of chemical structures active in vitro against the trypomastigote form of Trypanosoma cruzi

A rapid in vitro test system has shown that many drugs which possess a product licensed for use in man are also active at a concentration of less than 1mM against the blood forms of Trypanosoma cruzi. 62 of these are structurally related amphiphilic cationic drugs which completely lyse the trypomastigotes at 4 degrees C within 24 hr, yet most leave the erythrocytes intact. Three polyene and two anthracycline antibiotics were also found to be selectively trypanocidal under the same conditions.

Pyrimidine metabolism in Trichomonas vaginalis

Pyrimidine metabolism in Trichomonas vaginalis was investigated using washed cell suspensions of the organism with radiolabelled pyrimidine ring precursors and preformed pyrimidines. The precursors [14C]orotate, [14C]bicarbonate and [14C]aspartate were not incorporated into the pyrimidine bases of trichomonal nucleic acids, indicating that the protozoan is unable to synthesise the pyrimidine ring and is dependent on the salvage of exogenous pyrimidines. [3H]uracil, [3H]uridine, [3H]cytidine, deoxy[3H]cytidine and [3H]thymidine were all efficiently salvaged, and interconversion between cytosine and uracil nucleotides was detected. Thymidylate synthase activity was not detected, suggesting that T. vaginalis is dependent upon an exogenous supply of thymidine for TMP synthesis.

Enzymes of purine salvage in Trypanosoma cruzi

Previous experiments in which we examined the incorporation of radiolabelled bicarbonate, formate and glycine by washed cell suspensions of blood trypomastigote, intracellular amastigote and culture epimastigote forms of Trypanosoma cruzi suggested that no form of the organism could biosynthesize de novo significant amounts of purine [ 11. Absence of such biosynthesis implies dependence on salvage and in agreement with this, we detected substantial incorporation of radiolabelled purine bases and nucleosides, though not nucleotides [ 11. Here, we report the presence in cell-free homogenates of the three main forms of T. cruzi of phosphoribosyltransferase, aminohydrolase, kinase, hydrolase and phosphorylase activities which might be involved in this salvage. A preliminary report of some of the data has been presented [2].