Dihydrofolate reductases within the genus Trypanosoma

EXPERIMENTAL PARASITOLOGY 25, 311-318

Dihydrofolate

Reductases

J. J. Jaffe,2

(1969)

within

J. J. McCormack,

the Genus

Trypanosoma’

Jr., and W. E. Gutteridge

Medicine, The Zoological Society of London, Nufield Institute of Comparative London, N.W.1; Department of Pharmacology, The University of Vermont College of Medicine, Burlington, Vermont 05401; National Institute for Medical Research, Mill Hill, London N.W.7 (Submitted

for publication

4 November

1968)

JAFFE, J. J., MCCORMACK, J. J., JR., AND GUTTERIDGE, W. E. 1968. Dihydrofolate reductases within the genus Trypanosoma. Experimental Parasitology 25, 311318. Dihydrofolate reductase activity was detected in extracts of rat-adapted bloodstream forms of Typanosoma (Trypanozoon) brucei, T. (T.)rhodesiense, T. ( T.)equiperdum, T.( DuttoneZla)vivax, T. (Nannomonus) congolense (section Salivaria) ; T. ( Herpetosoma) lewisi, and T. ( Schizotypanum) cruzi ( section Stercoraria). This enzyme was also detected in extracts of culture forms of T.( T. )~hodesiense, T.( H.)lewisi, and T.( S.)cruzi. The trypanosomal dihydrofolate reductases shared the general properties of the analogous enzyme from diverse genera in their requirement for dihydrofolic acid as substrate and reduced nicotinamide adenine dinucleotide phosphate as cofactor. They also showed the characteristic extreme sensitivity to the inhibitory action of 4-amino analogs of folic acid such as methotrexate. On the other hand, the trypanosomal reductases exhibited a pattern of susceptibility to inhibition by certain 2,4diaminopyrimidines and related heterocyclic compounds which was different from the patterns of susceptibility to these agents that have been described for reductases from bacterial and mammalian sources. Superimposed upon this distinctive pattern, moreover, was a further differential response, whereby the drug sensitivities of the reductases of salivarian trypanosomes were almost identical and, as a group, clearly distinguishable from those of the stercorarian species. Those properties of dihydrofolate reductases from culture and bloodstream forms of T.( T. )rhodesiense, T.( H. ) Zewisi, or T. ( S. )cruzi that were compared were closely similar. INDEX DESCRIPTORS: Typanosoma spps; enzymes; dihydrofolate reductases; reequiperdum; ductase; Typanosoma brucei; Typanosoma rhooksiense; Trypanosoma Trypanosoma vivax; Trypanosoma congolense; Typanosoma lewisi; Typanosoma cruzi; 2,4-diamino-NlO-methylpteroyl glutamate ( methotrexate ) ; 2,4-diamino-5-pchlorophenyl-6-ethylpyrimidine ( pyrimethamine; Daraprim ) ; 2,4-diamino-5- ( 3’,4’, 5’-trimethoxybenzyl)pyrimidine (trimethoprim); 2,4-diamino-6-butylpyrido [2,3-d] pyrimidine; l-( p-butylphenyl)-l,2-dihydro-2,2-dimethyl-4,6-diamino-s-tr.

Species of mammalian trypanosomes have been primarily defined on the basis 1 This work was supported in part by Grant CA-08114-04 from the National Cancer Institute, U.S. Public Health Service, Bethesda, Maryland, and by Scheme No. R. 1975 from the Ministry of Overseas Development. 2 Present address: Department of Pharmacology, The University of Vermont College of Medicine, Burlington, Vermont 05401.

of morphology, development within the insect vector, and the nature of the infections they cause in susceptible hosts (Hoare, 1966; Ormerod, 1967). Accordingly, it has recently been proposed (Hoare, 1964, 1966) that the mammalian parasites I of the genus Trypanosoma be divided into two sections and seven subgenera. Section Stercoraria would include the subgenera 311

312

JAFFE, MCCORMACK,

Megatrypanum, Herpetosoma, and Schizotrypanum; section Salivaria, the subgenera Duttonella, Nannomonas, Pycnomonas, and Trypanozoon. Increasing knowledge concerning features of trypanosomal metabolism should prove useful in further clarifying distinctions among species and subgenera. The recent detection of the enzyme dihydrofolate reductase (EC 1.5.1.4) in two species, Trypanosoma WYPanoxoon) equiperdum and Trypanosoma (Schizotrypanum) cruzi (Jaffe and McCormack, 1967; Gutteridge and Senior, 1968), prompted this survey of available members of the genus for the presence and comparative properties of this particular protein. MATERIALS AND METHODS

The trypanosomal species studied were bloodstream forms of Trypanosoma (Trypanoxoon) equiperdum (obtained from the University of Vermont ) ; T. ( T.)brucei (strain Lister 427) and T.( Duttonella) vivax (obtained from the Lister Institute of Preventive Medicine, London) ; and T.( T.)rhodesien.~e (monomorphic strain), T. ( Nannomonas) congolense, T. ( Herpetosoma) lewisi, T. ( Schizotrypanum) and cruzi (Sonya strain) (all obtained from the National Institute for Medical Research, London). All species were ratadapted strains, with the exception of T. ( S. ) cruxi, which had to be grown in rats previously irradiated from a 6oCo source (610 rads/150-180 gm rat). Also available were culture forms of T. ( T. )rhodesiense (grown for us at the Microbiological Research Establishment, Porton, Hants.), T. (H.)Zewisi (grown for us at Mill Hill by Mr. B. Cover), and T.( S.)crud (grown by one of us, W. E. G. ) . Bloodstream form trypanosomes were separated from the other blood elements of infected rats by two different methods. For T. ( T. ) equiperdum, T. ( T. ) brucei, T.

JR., AND GU'I-lTRIDGE

(T.)rhodesiense, and T.( D.)vivax, in which the parasitemias in the rats were by high, separation was accomplished differential centrifugation, as described elsewhere (Jaffe, 1965). By this procedure, contamination with red and white blood cells was less than l%, but the proportion of platelets in the trypanosomal suspensions was generally around 20%. Consequently, suspensions of the rat platelets, equivalent in numbers to that encountered in the trypanosomal suspensions or greater by several logs of magnitude ( 1010-1013) were included as controls in the study. For T. ( N. ) congolense, T. ( H. ) lewisi, and T. (S.) cruzi, where the parasitemias were low, and with some preparations of T. (T. ) brucei, T.( T.)rhodesiense, and T.( D.) vivax, separation was accomplished with the aid of sucrose gradients after the blood had been defibrinated (F&on and Spooner, 1959; Williamson and Cover, 1966). These preparations were essentially free of both blood cells and platelets. Culture forms of T. ( T. )rhodesiense, T. (H. ) Eewisi, and T. ( S. )cruzi were harvested by centrifugation and then washed twice before use in calcium-free 0.116 M phosphate buffer, pH 7.6. Populations of 5 X log-5 X lOlo trypanosomes and 1010-1013 platelets were either subjected to acetone powdering (Jaffe and McCormack, 1967) and subsequently extracted with ice-cold O.lM phosphate buffer, pH 7.0, or they were resuspended in O.lM phosphate buffer, pH 7.0, containing 0.56% (w/v) N-acetylcysteine, disrupted by means of a Mickle disintegrator and the supernatant (after centrifugation at lo5 X g for 60 minutes) assayed directly. The method used to assay dihydrofolate reductase activity has previously been detailed (Jaffe and McCormack, 1967). Essentially it is based upon the decrease in absorbancy at 340 ml1 in the presence of dihydrofolic acid (prepared by the method

DIHYDROFOLATE

REDUCTASES OF TRYPANOSOMES

of Futterman, 1957) as substrate, the reduced form of nicotinamide adenine di(NADPH) as nucleotide phosphate hydrogen donor, and the enzyme. The assay was carried out in l-cm silica cells using either a Beckman DU spectrophotometer equipped with thermospacers to maintain the cell compartment at 37°C or a Unicam SP 700C recording spectrophotometer equipped with a thermostatically controlled cell compartment. One unit of enzyme is defined as that quantity of protein which catalyzes the reduction of 1 mumole of dihydrofolate per minute and was estimated by the method of Blakley and McDougall (1961). Protein was measured according to the method of Lowry et ~2. (1951). The method used to assay lactic acid dehydrogenase activity was essentially that of Markert and Appella ( 1961). Methotrexate was obtained from Lederle Laboratories, Pearl River, New York. Dr. M. Weatherall, Wellcome Research LaborWellcome and Co., atories, Burroughs Beckenham, Kent, kindly provided the following 2,4-diaminoheterocyclic compounds: BW 50-63: 2,4-diamino&p-chlorophenyl-6ethylpyrimidine ( pyrimethamine); BW 5672: 2,4-diamino-5- (3’,4’,5’-trimethoxybenzyl) -pyrimidine ( trimethoprim ) ; BW 60212: 2,4-diamino-6-butylpyrido [2,3-d] pyrimidine; BW 5743: l- ( p-butylphenyl) -1,2dihydro-2,2-dimethyl-4,6-diamino-s-triazine. RESULTS

Sources of Trypanosomul Dihydrofolate Reductases Dihydrofolate reductase activity was detected in extracts of all four species separated from the blood of infected rats by ( T. ( T. ) epiperdifferential centrifugation dum, T. ( T. ) brucei, T. ( T. ) rhodesiense, and T.( D.)viuux). On the other hand, there was no detectable dihydrofolate reductase

313

activity in extracts of rat platelets a thousand times more abundant than the numbers that were calculated to be present in trypanoour suspensions of blood-form somes. In contrast, the lactic acid dehydrogenase (LDH, EC 1.1.1.27) activity in such extracts of platelets was high (in the order of 1.1.5 umoles pyruvate reduced per milligram protein per minute), and we found that it could account for the total LDH activity in extracts of bloodstream form T.( T.)equiperdum separated by differenthus supporting the tial centrifugation, finding of Dixon (1966) who studied bloodstream form T.( T.)rhodesiense separated in like manner. The negligible condihydrofolate reductase tribution of activity by rat platelets suggested that we could ignore these contaminants of some of our trypanosomal suspensions insofar as this survey was concerned. Our later observations that extracts of T. ( T.)rhodesiense, T. ( T. ) brucei, and T. ( D. ) vium, prepared essentially free of platelets by the use of a defibrinating technique coupled with sucrose gradients, had dihydrofolate reductase activity virtually identical with those in platelet-contaminated extracts confirmed our suggestion. Dihydrofolate reductase activity was also demonstrated in pure preparations of bloodstream forms of T.( N.)congoZen.se, T.(H.)bwisi, T.( S.)cruzi, and in preparations of culture forms of T. ( T. ) rhodesiense, T.(H.)lewki, and T.(S.)cruzi. We thus demonstrated such activity in seven species, representing the five most important subgenera of the genus Trypanosoma. The specific enzymic activity in the crude extracts from all sources except the culture form of T.( S.)cruzi generally ranged from 10 to 20 units per milligram protein. Some variation in specific enzymic activity was observed in different preparations of a given species, possibly due to variable efficiency of the extractive procedure. However, it is noteworthy that the

314

JAFFE,

MCCORMACK,

specific activity of dihydrofolate reductase in crude extracts of culture form T.( S.) C?YLZi was consistently much higher (around 90-100 units mg protein) than that in the other extracts, including those of bloodstream form T. (S.) cruzi.

JR., AND GUTTERID~E TABLE I (K,) of Dihydrofolic Acid (Substrate) and NADPH (Cofactor) for Dihydrofolute Reductases of Diverse Origin

Afinities

K wk Dihydrofolic acid M x 10-G

Species

Substrate and Cofactor Requirements When an equimolar amount of folic acid was substituted for dihydrofolic acid in the standard assay system for each of the trypanosomal extracts, or when the reduced form of nicotinamide adenine dinucleotide (NADH) replaced NADPH, very little decrease in absorbancy at 340 rnp was observed, indicating the superiority of dihydrofolic acid as substrate and NADPH as hydrogen donor in the reaction mediated by trypanosomal dihydrofolate reductases. Such properties are characteristic of this enzyme from whatever source. The apparent affinity constants (K,) for dihydrofolate and NADPH were determined for most, but not all, of the trypanosomal reductases, since the availability of some material was verv limited. The values appear in Table I, together with those we determined for closely related crithidial reductases, those for the reductase from a representative of the Eubacteriaceae, (Burchall and Hitchings, 1965) and the K, of dihydrofolate for human reductase (Burchall and Hitchings, 1965) for the purpose of comparison. The K, values for dihydrofolate obtained for the trypanosomal reductases ranged from 3 x lo-OA4 to 1 x 10-5M, well within the extremes for this parameter which appear in the literature (Jaffe and McCormack, 1967). The K, values for NADPH, all around 1 x lo-“M, were also closely similar to those determined for dihydrofolate reductases of other genera. The data further indicate that, with the possible exception of T. ( H. ) lewisi, there were no

T. f T. bbrucei% T. (T.) rhodesienseo T. (T.) rhodetiense” T. (T.) equiperduma T. (D.) vivu~a T. (N.) congolensea T. (H.) lewisia T. (H.) Zewisib T. (S.) cm& T. (S.) cruzib C. fasciculutab C. oncopeltib E. colic Mane ~

I

Km NADPH a4 x 10-G

3

-

8

14 12 10 -

5 4 7 3 10 3 4 8 3 3 26

(1 Bloodstream form. b Culture form. C Data of Burchall and Hitchings

5

9 9 11 12 10 -

(1965).

marked differences in affinity of substrate and cofactor for the reductases from culture forms and bloodstream forms of the same species. Znhibition

Studies

The trypanosomal reductases, like those from other genera, were strongly inhibited by the 4-amino analog of folic acid, methotrexate. For example, the concentration of methotrexate required for 50% inhibition ( IDno) ranged from 2 X lo-lo (T.( T.) equiperdum) to 3~ 10-sM (T.(T.) rhodesiense), culture form (Table II). Burchall and Hitchings (1965) found that dihydrofolate reductases from mammalian sources on the one hand, and from bacterial sources on the other exhibited two markedly dissimilar patterns of sensitivity to the inhibitory action of various 2,4-diaminopyrimidines and related hetero-

DIHYDROFOLATE

REDUCTASES

TABLE

OF

315

TRYPANOSOMES

II

Comparative Sensitivities of Dihydrofolute Reductases to Inhibition by Diaminoheterocyclic Antimetabolites Concentration Snecies

T. (T.) bruceif T. (T.) rhodesiensef

T. T. T. T. T. T. T. T. E.

(T.) rhodesienseg (T.) equiperdwnf (D.) vivad (IV.) congoknsef (H.) lewisi-f (H.) Zewkig (S.) cruzit (S.) cruzis

co@ Ma& a b 0 d e f g *

(X 108 M) for 50% inhibition

MTXQ

PYRb

TRIG

60-212d

57-43e

0.08 0.10 0.30 0.02 0.07 0.07 0.20 0.10 0.15 0.10 0.10 0.20

20 20 40 20 25 25 750 500 100 200 250 180

50 25 75 100 70 70 2000 2000 1000 1000 0.5 30000

50 60 70 40 40 60 80 70 30 70 50 95

700 400 700 2000 300 400 6000 600 300 500 65000 55

MTX (methotrexate): 2,4-diamino-Nlo-methylpteroyl glutamate. PYR (pyrimethamine): 2,4-diamino-5-p-chlorophenyl-6-ethylpyrimidine. TRI (trimethoprim): 2,4-diamino-5-(3’,4’,5’-trimethoxybenzyl) pyrimidine. 6&212 (BW60-212): 2,4-diamino-6-butylpyrido [2,3-d] pyrimidine. 57-43 (BW.57-43): l-(p-butylphenyl)-l,2-dihydro-2,2-dime~yl-4,6-diamino-s-t~azine. Bloodstream form. Culture form. Data of Burchall and Hitchings (1965).

cyclic compounds. We found that the analogous trypanosomal reductases were also inhibited by this class of compounds, but that they, as a group, showed yet a third distinctive pattern of sensitivity to these agents, particularly to trimethoprim and to the diaminotriazine, BW 5743 (Table 11). These latter two compounds possess the most striking ability to discriminate between dihydrofolate reductases of bacterial and mammalian origin. The sensitivity of all trypanosomal reductases to their action clearly fell between those extremes. At the same time, it was observed that the reductases from T. ( T. )brucei, T. T. ( T. ) rhodesiense, T.( T.)equiperdum, and T. (N. ) congolense had CD.) vivax, closely similar drug-sensitivity profiles, clearly distinguishable in certain respects from those of T.( H.)kwisi and T.( S.) cruzi. The reductases of T. (H.)lewti and

T.( S.)cruxi showed similar sensitivity to trimethoprim, both requiring around 20 times more drug on a molar basis than the other trypanosomal reductases to be inhibited by 50%. The sensitivity of the lewisi and cruzi reductases to pyrimethamine was more variable but still distinctly lower than that of the other trypanosomal reductases. It is noteworthy that a third 2, 4-diaminopyrimidine derivative, BW 60212, previously shown by Burchall and Hitchings ( 1965) to exert a uniform action against dihydrofolate reductases of diverse origins, had the same undiscriminating action against the trypanosomal reductases. The drug-sensitivity profiles of dihydrofolate reductases from culture and bloodstream forms of the same species (either T. ( T. )rhodesiense, T. ( H. ) lewisi, or T. ( S. ) cruzi) were closely similar, except for the lo-fold greater sensitivity of the enzyme

316

JAFFE,

MC CORMACK,

from culture form T. (H.)Zewisi over that of the enzyme from the bloodstream form of this species to the action of BW 57-43. DISCUSSION

Dihydrofolate reductase activity has now been detected in extracts of seven species two subof trypanosomes, representing genera of the section Stercoraria and three subgenera of the section Salivaria. Previously, a substance (or substances) was of T.(T.) isolated from homogenates equiperdum which possessed properties closely resembling those of folic acid or a 2-amino-4-hydroxyprelated conjugated teridine ( Jaffe and McCormack, 1967). The unconjugated 2-amino-4-hydroxypteridine, biopterin, was neither an effective substrate nor an inhibitor of dihydrofolate ( Mcreductase from T. ( T. ) equiperdum Cormack and Jaffe, 1967), and more recently it was found that dihydrobiopterin (Kaufman, 1967) was similarIy inert (unpublished data). This evidence, taken together, strongly suggests that members of the genus Trypanosoma have a normal folate metabolism, whereby dihydrofolic acid is converted to the biologically active tetrahydro form in the presence of dihydrofolate reductase and NADPH. We have also detected dihydrofolate reductase activity in extracts of both Crithidia faxicuZata and Crithidia oncopelti (cf. Table I), further details concerning which will be published later. In view of the latter findings, the role of biopterin (“crithidial factor” of Nathan and Cowperthwaite, 1955) in crithidial metabolism should be further examined. Our studies indicate that while all the trypanosomal dihydrofolate reductases share the general properties of the analogous enzyme extracted from cells of diverse genera, they exhibit, as a group, a of susceptibility to distinctive pattern inhibition by certain 2,4-diaminohetero-

JR.,

AND

CUTTJZRIDCE

cyclic antimetabolites. The usefulness of such chemical agents to reveal intragroup specificities of dihydrofolate reductases has already been well documented (Baker and Ho, 1964; Burchall and Hitchings, 1965; Hitchings and Burchall, 1966). Superimposed upon this distinctive “inhibitor profile” of the trypanosomal reductases, however, was a further differential response, for it was found that the reductases of T.( T.)brucei, T.( T.)rhodf&nse, T.( D.)vivax, and T. T. ( T.) equiperdum, ( N. ) congobnse had closely similar drugsensitivity profiles, clearly distinguishable in certain respects from those of the reductases of T.( H.)Zewisi and T.( S.)cruzi. One might expect expect that the dihydrofolate reductases of T. ( T. ) brucei and T. ( T. )rhodesiense would be almost identical, conpresented by sidering the evidence Ormerod (1967) to support his proposal that T. brucei, T. rhodesiense, and T. gambien.se be synonymous with T. (T. ) brucei. But it is quite remarkable that the reductases of T.( T.)equiperdum, T.( D.)vivax, and T. (N. )congolense, species with such disparate morphology, epidemiology, and clinical pattern, should also be mutually indistinguishable. Such similarities and differences in properties between a particular protein of salivarian and stercorarian trypanosomes may reflect phylogenetic kinship and divergencies. Hoare (1948) and Baker (1963) have suggested that the members of the T. brucei, T. oivax, and T. congolen-se subgroups (the older classification of Hoare, 1949, 1957) had a common ancestor, had evolved on the African continent relatively recently, and have a number of shared features which tend to separate them from the majority of species of the genus. In his studies of phospholipids in trypanosomes, Godfrey (1967) observed that T. ( H. ) Zewisi, representing the section Stercoraria, had considerably less sphingomyelin and more lecithin than the salivarian species, T. ( T. ) brucei, T.

DIHYDROFOLATE

REDUCTASES

( D. ) uiuux, and T. ( IV.) congolmse. In view of our findings, it would be interesting to compare the properties of dihydrofolate reductases of more members of the genus, especially those species which parasitize aquatic vertebrates and are transmitted by leeches. Perhaps this biochemical tool could prove useful in future studies concerned with the origins of the African trypanosomes in particular and the phylogenetic relationships of the genus as a whole. The finding that the dihydrofolate reductases of both culture and bloodstream forms of a given species were closely similar insofar as the parameters of comparison in this study were concerned deserves further comment. It is well known that in most cases the physiology and metabolism of these two developmental forms differ profoundIy (Pizzi and Taliaferro, 1960; Ryley, 1962; Vickerman, 1962). Yet important structural features of a key enzyme of folate metabolism apparently remain little affected by marked shifts in the environment, implying that the binding sites of dihydrofolate reductase are unaltered during the differentiation of a given species of trypanosome in the course of its life cycle. That the properties of culture form reductases of T. ( T. )rhodesiense and T. ( S. )cruzi were found to be closely similar to those of their counterparts in bloodstream forms has a practical advantage. Culture forms of these species can be grown in large batches and the massive populations that are desirable for biochemical processing can thus be obtained more conveniently than is possible with bloodstream forms. Further comparative studies of analogous enzymes derived from these representatives of the sections Salivaria and Stercoraria are contemplated. Finally, we should like to call attention to the finding that the dihydrofolate reductases of the African trypanosomes were 300-1000 times more sensitive to the inhibitory action of the 2,4-diaminopyrimi-

OF

TRYPANOSOMES

317

than was the human dine, trimethoprim, dihydrofolate reductase (as determined by Burchall and Hitchings, 1965). Theoretically, such a difference in sensitivity of the analogous enzyme in parasite and host offers an opportunity for exploitation by chemotherapy. In practice, neither trimethoprim nor indeed any presently available dihydrofolate reductase inhibitor has exerted effective antitrypanosomal action in viuo. We have some evidence to suggest that this therapeutic failure may in part be due to such pharmacodynamic factors as degree of plasma protein binding and poor penetration into the parasites. Whatever the reasons may turn out to be, we feel that dihydrofolate reductase as a target for chemotherapy is too promising to be ignored. ACKNOWLEDGMENTS We gratefully acknowledge our indebtedness to the Wellcome Trust and the Wellcome Foundation, Ltd. for Research Travel Grants which enabled two of us (J. J. J. and J. J. M.) to be in London for the collaborative effort which led to this report. The excellent technical assistance of Mrs. Frieda Claes, Miss Jacqueline O’Brien, Miss Jane Dunnett, and particularly Mr. Bryan Cover is recognized with thanks. We also thank Drs. L. G. Goodwin, C. E. Gordon Smith, and G. H. Hitchings for their interest in this project and the material assistance they provided for its undertaking.

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