Folate Metabolism in Malaria
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Bulletin of the World Health Organization, 55 (2-3): 291-298 (1977) Folate metabolism in malaria ROBERT FERONE 1 It is known that malaria parasites are inhibited by sulfonamides and antifolate com- pounds, require 4-aminobenzoic acid for growth, and respond only partly to intact folic and folinic acids. Biochemical data obtained during the last decade on the synthesis of nucleic acid precursors and on folate enzymes in malaria support the hypothesis that malaria parasites are similar to microorganisms that synthesize folate cofactors de novo. Sulfa drugs inhibit plasmodial dihydropteroate synthase (EC 2.5.1.15). Pyrimethamine and many other antifolate compounds bind to tetrahydrofolate dehydrogenase (EC 1.5.1.3) of the parasite more tightly than to the host enzyme. However, the metabolic consequences of the depletion offolate cofactors as a result of drug inhibition are not yet known. Other areas to be studied are the origin of the pteridine moiety offolates, the addition ofgluta- mate (s) in folate cofactor biosynthesis, the means by which intact, exogenous folates affect malarial growth, and demonstration of the enzymes and reactions involving N5- methyl tetrahydrofolate. Studies on the sulfonamides and 4-aminobenzoic DIHYDROFOLATE BIOSYNTHESIS a acid (pABA) in malaria date back about four The growth of plasmodia is greatly stimulated by decades, when sulfachrysoidine and sulfanilamide pABA, in vivo and in vitro. The growth of P. know- were first used to treat human malarial infections (1) lesi in the Harvard medium developed during World and pABA was found to reverse sulfonamide inhibi- War II (4) required pABA, which prevented in- tion in Plasmodium gallinaceum (2). Since then, there hibition of growth by sulfadiazine (5). Erythrocytic have been many reports of studies on the effects of forms of several species of plasmodium grew poorly folate precursors, folate metabolites, folate end- or not at all in hosts fed on pABA-deficient diets. products, and folate analogues on malarial growth Hawking (6) reported the suppression of parasitaemia and survival in vivo and in vitro. Most of these data of P. berghei in the rat and P. cynomolgi in the rhesus are consistent with the current general view of folate monkey fed on milk diets, which were shown to metabolism in most bacteria; these organisms have a low pABA content. In both cases, pABA synthesize the needed folate cofactors de novo and do supplementation of the milk diet allowed the infec- not utilize the exogenously supplied, intact folate tions to develop. Other reports followed, with varying molecule (3). The enzymatic evidence accumulated results, including some sulfonamide-resistant strains in the last decade supports this view, so that the of P. berghei that grew well in mice on a milk diet general outline of the origin of folate cofactors -see Peters (7) for a review. Some of the variability within the malarial parasite seems to be established. observed could be due to differences between strains However, there are still questions to be answered on in their capacity to utilize low levels of pABA, the details of the enzymes involved, on the functions since Vray (8) found that, in mice on a pABA-defi- of folate cofactors in malaria, on the mechanism by cient diet, blood levels of pABA fell rapidly but did which exogenous folates exert their effects in mal- not completely disappear. The pABA level in the aria, and on the physiological consequences of the liver remained high, allowing normal development inhibition of folate cofactor biosynthesis by sul- of hepatic schizonts after sporozoite inoculation. fonamides and antifolate drugs. This article reviews what is known and what questions remain unan- a Abbreviations used: pABA (4-aminobenzoic acid); in malaria. pABG (4-aminobenzoylglutamate); H,-folate (7,8-dihy- swered on folate metabolism dropteroylglutamate); H.pteroate (7,8-dihydropteroate) ;pt (2- amino-4-hydroxy-pteridine); H.ptCH,OH (6-hydroxymethyl- 1 Senior Research Microbiologist, Department of Micro- 7,8-dihydro-pt); H.ptCH,OPP (6-pyrophosphorylmethyl-7,8- biology, Wellcome Research Laboratories, Research Triangle dihydro-pt); H,-folate (5,6,7,8-tetrahydropteroylglutamate); Park, NC 27709, USA. folinic acid (N'-formyl-H,-folate). 3608 - 291- 292 R. FERONE The pABA requirement of P. falciparum has been the sulfonamides in bacteria. The enzyme has been indicated by studies of Kretschmar, who suggested found in extracts of P. chabaudi (14), P. berghei (15, that the low incidence of P. falciparum in children 16), and probably P. knowlesi, P. lophurae, and fed on milk diets was due to the low content of P. gallinaceum (15). The three last-mentioned species pABA in milk (9). Kretschmar & Voller (10) later were assayed with pABG in place of pABA as showed that P. falciparum did not grow in Aotus substrate; pABG has been shown to be an alternative monkeys maintained on a milk diet. The experiment substrate to pABA in bacteria and plants (13) and would have been more conclusive, however, had it P. berghei (15). demonstrated that the parasite would grow if pABA The enzyme before dihydropteroate synthase in were included in the milk diet. the pathway of H2folate biosynthesis is 2-amino-4- Jacobs (11) published a detailed study and dis- hydroxy-6-hydroxymethyldihydropteridine pyro- cussion of the role of pABA in malarial infections, phosphokinase (EC 2.7.6.3) (Fig. 1). In Escherichia in vivo. He observed increasing parasitaemia in coli these two enzymes are separated by DEAE- mice infected with P. berghei (NYU-2 strain) with cellulose or Sephadex G-100 chromatography (13). increasing pABA levels in the diet, and no growth In Lactobacillus plantarum there was evidence both in its absence. A pyrimethamine-resistant strain for a complex and for a separate dihydropteroate required higher levels of pABA for optimum de- synthase from a DEAE-cellulose column, (17), and velopment. Folic and folinic acids only partially in pea seedlings the two activities resided in a single replaced pABA. Jacobs concluded that "... the protein (18). I found that these two enzymatic activ- parasite is unable to utilize the preformed folic or ities coeluted when a P. berghei crude extract was folinic acid molecule and that pABA is used by the run on a Sephadex G-200 column, but they could be parasite to form a substance similar to, but not separated by DEAE-Sephadex chromatography (15). identical with, folic acid." (11). On the basis of our Walter & Konigk reported that they could not sepa- current knowledge on folate biosynthesis, that rate the synthase and pyrophosphokinase activities substance would appear to be dihydrofolate. from P. chabaudi during a purification scheme that The competitive nature of the reversal by pABA of resulted in a 950-fold increase in specific activity (19). sulfonamide inhibition has been documented in However, they did not use an assay that would have several species of plasmodia (7, 12). By analogy detected the pyrophosphokinase activity alone, in the with the metabolic pathways established in bacteria, crude extracts or early fractions. The combined assay this should mean that the sulfa drugs compete with used determines the specific activity of the limiting pABA for binding to the enzyme dihydropteroate reaction only, and, if a separate pyrophosphokinase synthase (EC 2.5.1.15) (3). This enzyme catalyses were present in excess of the dihydropteroate syn- the condensation of pABA with H2ptCH2OPP to thase specific activity, it would not have been detected. form H2 pteroate (Fig. 1) and is the site of action of The protein fraction they isolated carried out the combined reaction, and the two activities were found together on Sephadex column chromatography of a QH OH P. berghei crude extract, so it is likely that complexes N-N N- N-1-CH20H ofthese enzymatic activities exist in rodent plasmodia. iN R-P-P-PR-P-P-PH2N NHH Several other differences were found for these en- in (GTP) (H2 Pt-CH20H zymes the studies reported (Table 1), which may be due to species differences, the presence or absence ATP H2Pt-CH20H of the enzyme complex in the fractions studied, mg Pyrophosphokinose or differences in methodology. Most importantly H2N COOH 4, though, two key enzymatic reactions required for p-ABA OH ~~~~~~~~~~~~N dihydrofolate biosynthesis have been demonstrated N H H ZwCH20PP N- 1<1C-N C~OOH .I> in malarial parasites. H H2 Pteroote H N H H Sn "2N N etsH The inhibition of malarial dihydropteroate syn- (H2 - Pteroote) (H2Pt-CH2OPP) thase by sulfonamides and dapsone and their com- petition with pABA have also been reported (Table 1). Fig. 1. Synthesis of H2pteroate by bacteria and plants The constants obtained are (13), showing the reactions of 2-amino-4-hydroxy-6- inhibition (Ki values) hydroxymethyidihydropteridine pyrophosphokinase (EC comparable to these determined with the enzyme 2.7.6.3) and dihydropteroate synthase (EC 2.5.1.15). from bacterial sources (13) and satisfactorily con- FOLATE METABOLISM IN MALARIA 293 Table 1. Properties of malarial dihydropteroate synthases Sp. act. pH Apparent Km values (Mmol/litre) Apparent Ki values (pmol/litre) Refe- (pmol/ Source Substrate rence opti-mum H2ptCH20H H2ptCH20PP pABA pABG DDS sulfa- sulfa- sulfanil- protein)min/mg diazine thiazole amide P. H2ptCH20H a (15) 1 7.0 0.8 - 0.28 37 0.38 1.4 b 0.56 25.2 berghei H2ptCH20PP (21) 1 ND c - 1.4 0.21 ND ND ND ND ND P. H2ptCH20PP (16) 2 8.5 - 1.4 2.8 ND 19 b 39 b ND ND berghei P. H2ptCH20H (19) 16.5 8.7 11.0 - 1.5 ND ND ND 1.4 13.0 chabaudi H2ptCH20PP (19) 16.0 8.7 - ND ND ND ND ND ND ND a Combined assay method for malarial 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase and dihydropteroate synthase.