Pteridine synthesis in malaria parasites

Parasitology Today, voL 2, no. 3, 1986

57

lessen the impact of disease have not been developed. ~ssueof the Amer/can oyster. Three nuclei with eccentric endosomes and K ~ (m~crorubules of mito~ apparatus) are visible in the plasmodium.

Other Parasites There are numerous other parasites of shellfish including viruses, bacteria, ciliates, trematodes, zooflagellates, amoebae and copepeds15. However, none has been of much importance except for the copepod, Myg~ola or/ental/s, and the facultative, bacterial pathogens (Vibyiospp.), the latter of which cause severe mortalities of larval bivalves in hatcheries with inadequate cleanliness. Shellfish Farming Shellfish farming is best conducted with local stocks, because of the danger of introducing parasites which can become highly virulent in their new environments. Although proof is often lacking, it is strongly suspected that major epizoofics have been induced by moving sheUfish from their natural interbreeding populations to other sites. T h e distances need not be great, H. nelsoni was introduced into Massachusetts waters probably from less than 200 miles south. T h e importation of C. #gas into French waters has not been proven to be responsible for the severe epizootics caused by M . refringens a n d B. ostreae, but the suspicion remains, despite the fact t h a t C . g/gas is resistant to the disease. Once a parasite becomes established, the shellfish farmer needs help to see if salinityinduced refuges can be found, and to determine the life cycle of the parasite so that the impact of infection can be lessened by planting earlier or later in the year or by

harvesting when the molluscs are smaller. Where intertidal culture occurs the height of oyster culture should also be considered as a means of lessening the impact of disease s .

NEw5

Rd~n~ 1 Ray, S.M. (1954) The Rice Institute Pamphlet, Special IssueHouston,Texas 2 DaRo%L. and Canzonier,W.J. (1985)Bull. Eur. Assoc. Fish Parkol. 5, 23-27 3 Perkins,F.O. (1985)Prograntr~a, dAbsrractsoftheVll Imerna~mal CongressofProtozoologyp. 81, 22-29June, 1985,Nairobi,Kenya 4 Andrews,J.D. and Hewatt,W.G.(1957)Ecol. Menogr. 27, 1-26 5 Mackin,J.G. (1962)Publicarioasofthel~ofMarine Science of the Universityof Texas 7, 132-229 6 Lester, R.J.G. and Davis, G.H.G. (1981)J. Invert. Pathol. 37, 181-187 7 Grizel,H. (1985)Etudes des recentesepizootiesde rhuitre p/ateOareaedu2islinn~a de/eur/mpartsur l'ostre/odmre bretomwThesis,Acad.Montpellier,Univ.Sci.Techniq. Lanquedoc. 145pp. 8 Wolf,P.H. (1979)US NationaIMmim FisheriesService MarineFisher/esRev~o 41, 70-72 9 Andrews.J.D. (1966)Ecology45, 19-31 10 Andrews,J.D., Wood,J.L. and Hoese, H.D. (1962) J. Insect. Pazhol. 4, 327-343 11 Haskin, H.H. and Ford, S.E. (1979) US National Marine Fisheries Service Marine Fisheries Rev/ew 41, 54-63 12 Farley,A. (1967)J. Protozool. 14, 616-625 13 Pichot, V. a al. (1979) Rev. Travaux Inst. Peches Mar/6mes43, 131-140 14 Alderman,D.J. (1976)in Recent Advances in Aquatic Mycology(E.B. GarethJones, ed.) pp. 230-260, Elek Science,London 15 Sindermaun,C.J. (1970) Principal Diseases of Marine Fish and Shellfish, AcademicPress,NY

Pteridine Synthesis in Malaria Parasites D.C. Warhurst

Purines and pyrimidines are essential building blocks for nucleic acids. Unlike their mammalian host, malaria parasites lack the pathways necessary to use preformed pyrimidines. They synthesize pyrimidine nucleotides from simpler precursors, one of the crucial steps being the transfer of a methyl group from methylated tetrahydrofolate cofactor to uridy-

late in the thymidylate synthetase reaction. Malaria parasites, like other microorganisms, synthesize their folate cofactors from pteridine, 4-aminobenzoic acid (PABA) and glutamate. Like glutamate, PABA is derived from the diet of the host, and malaria is known not to thrive in animals on a PABA-deficient diet such as milk. This dependence on PABA means

that plasmodial growth is inhibited by the sulphonamides, which are PABA analogues. In addition, drugs such as pyrimethamine, which specifically inhibit plasmodial dihydrofolate reductase (the enzyme responsible for the reduction of dihydrofolate to tetrahydrofolate) are potent antimalarials, even though they act relatively slowly. Because sulphonamide

~)1986, ElsevierSciencePublishersBY, Amsterdam 01694758/86/$0200

58

Parasitology Today, vol. Z no. 3, 1986

action tends to reduce the synthesis ofdihydrofolate (Fig. I), combinations of sulphonamide and dihydrofolate reductase inhibitors show potentiative synergism.

combined with PABA in the first stage of folate synthesis. The second of these enzymes, a pyrophosphokinase, has already been found in malaria 3. Krungkrai et al. grew the human malaria parasite, Plasmodium falciparum (KI strain, resistant to chloroquine, pyrimethamine and sulphadoxine) in culture, removed the host erythrocytes by saponin lysisand then lysed the washed parasites by freeze thawing. The high-speed supernatant of the lysate was initially assayed for the GTPCase by following the production of D-erythro-dihyd roneopterin triphosphate from GTP and also the release of i4C-formate from 8- f4C-GTP. The enzyme was purified 200-fold from lysates by high performance sizeexclusion chromatography (HPSEC) and had a relative molecular weight of 300 kDa. It was significantly inhibited by ATP (Ki = 600 I~M) and had a K m for GTP of 54.6 I~M. An inhibitor of GTPCase, N 7-methyl guanosine, had aK, of 4.2 I~M, and

Newly DiscoveredEnzyme Pteridines have generally been assumed to be synthesized by the parasite from guanosine triphosphate (GTP) as in bacterial A paper recently published in Molecular and Biochemical Parasitology by Krungkrai et al., from a joint Thailand-USA research collaboration 2, is the first report of the occurrence in malaria parasites of the necessary enzyme, guanosine triphosphate cyclohydrolase (GTPCase). This enzyme splits off formate from guanosine triphosphate (GTP) and converts the product to the pteridine, D-erythrodihydroroneopterin triphosphate. This compound needs to undergo two further enzymatic transformations, before being Fig. I Synthesisof DNA from guanosine triphosphate (GTP) by malaria parasites. Starred enzymes have been detected in malaria parasites, others ore presumptive. $ ~ indicatessite of action of sulphonamides such as sulphadoxine; $2 indicatessite of action of pyrimethamine and cycloguaniL PABA

GTPCase*

dihyclroneopterin triphosphate

I

I

dihydroneopterin aldolase

I 2-amino-4-hydroxy

I

6-hydroxymethyl I dihydropteridine |

> dihydropteroate * synthetase

dihydropteroate ~lutamaCe dihydrofolate synthetose

I

dihydrofolate

I

dihydrofolate* reductase

J tetrahydrofolate I serine

I

serine hydroxymethyJ-* -transferose

N--S,N-10-mmhylene

)

tetrghydrofolate

I

thymidylate

I

~

~

I :b I I I ]

was shown to have antimalarial activity in vitro with a 50% inhibition concentration (ICs0) of 160 HM. Having characterized the enzyme from P. falciparum, the authors studied its occurrence in P. berghei, P. knowlesi and P. falciparum from an infected patient. Host red cells showed no activity. In synchronized P. knowlesi infections it was shown that the specific activity of the enzyme increased from the ring to the trophozoite stage, but decreased in the schizont, similar to glutamate dehydrogenase. GTPCase has not previously been identified in parasitic protozoa, its presence in plasmodia suggests the existence of a pteridine synthetic pathway similar to that described in bacteria. Comparison of the characteristics of the plasmodial enzyme with that isolated from other organisms is instructive. The molecular weight is in the high range, between, that of Escherichia coil (210 kDa) and Drosophila (345 kDa) (mammalian and avian enzymes are around 125 kDa). The enzyme is not dependent on magnesium, which makes it similar to that from E. coil Although the K i value for ATP is high, the intracellular concentration in infected cells is relatively high, and Krungkrai et aL suggest that ATP may play a regulatory role in the intact parasite. Radioactivity from labelled GTP was incorporated into dihydroneopterin, neopterin and folylpolyglutamate in P. falciparum, and the labelling of the last compound was inhibited by sulphadoxine, linking the synthesis of the pteridine to biosynthesis of folate. Although the microbial pathway of folate synthesis is clearly of major importance to plasmodia, there is some evidence that the parasites can salvage and use preformed folate from the host, because folate added in vitro can antagonize the action of sulphonamide drugs4. The authors point out that de nova synthesis of folate cofactors is a parasite specific metabolic pathway, and targetting new antimalarial agents ag~nst GTPCase could be a new area for chemotherapeutic studies. Comparison of the Ki of N - 7 methyl guanosine for the isolated enzyme of 4.2 pM, with the IC50in vitro of 160 I~M, suggests that there may be permeability barriers to overcome, or it may be that the free availability of GTP in the cell is a stumbling block. Nevertheless the differences between the host and parasite enzyme certainly indicate scope for chemotherapeutic differentiation.

syn~etase*

RefarL~ deoxythymidylate

I Ferone, H (1977) Bull. WHO 55, 291-298 2 Krungkrai, J., Yuthavong, Y. and Webster, H.K. (1985) MoL Biochem. Parasitol. 17, 265-276 3 Ferone, H (P973)J. Protozaol. 20, 459-464 4 Chulay, J,D., Watkins, W.M, and Sixsmith, D.G. (1984 ) Am. J. Trap. Med. Hyg. 33, 325-330