The gene for hypoxanthine phosphoribosyl transferase of Plasmodium falciparum complements a bacterial HPT mutation

Molecular and Biochemical Parasitology, 41 (1990) 281-288 Elsevier

281

MOLBIO 01364

The gene for hypoxanthine phosphoribosyl transferase of Plasmodium falciparum complements a bacterial HPT mutation M o h a m m e d S h a h a b u d d i n a n d J o h n Scaife Department of Molecular Biology, University of Edinburgh, Edinburgh, U.K. (Received 28 November 1989; accepted 19 March 1990)

The enzyme hypoxanthine phosphoribosyl transferase of Plasmodiumfalciparum has been overexpressed in Escherichia coli. The protein was found to be active enzymatieally. When the recombinant expression vector (pPfPRT2) was transformed and expressed in a Salmonella typhimurium mutant KP1684 (pure deoD hpt gpt), the active expressed protein complemented the hpt mutation in the bacteria. We discuss the practical value of this strain. Assays of the expressed protein in the mutant extract showed that the enzyme is able to use hypoxanthine, guanine and xanthine as substrates. A specificity study using the competitive inhibitor, 6-thioguanine, showed that of these hypoxanthine is the most favourable substrate. The biological significance of xanthine utilisation by the enzyme is discussed. Key words: Complementation; Hypoxanthine phosphoribosyl transferase; Malaria; Plasmodium; Purine metabolism

Introduction

The human malaria parasite, Plasmodium falciparum, like many other parasitic protozoa is unable to synthesise the purine ring de novo [1]. Instead, it scavenges them from the host. There is an enzyme which serves this purpose in P. falciparum [2,3]. This is hypoxanthine phosphoribosyl transferase (HPRT; IMP: pyrophosphate phosphoribosyl transferase EC. 2.4.2.8). The parasite obtains hypoxanthine from the host and converts it using this enzyme into inosine monophosphate Correspondence address: John Scaife, Dept. of Molecular Biology, University of Edinburgh, King's Buildings, Mayfield Road, Edinburgh EH9 3JR, Scotland, U.K. Abbreviations: AICAR, phosphoribosyl-5-amino-4-imidazoleearboxamide; AIR, phosphoribosyl-5-aminoimidazole; BSA, bovine serum albumin; CAIR, phosphoribosyl-4-earboxy-5-amlnoimidazole; GPRT, guanine PRT; gpt, bacterial gene for GPRT; HPRT, hypoxanthine PRT; hprt, Plasmodium falciparum gene for I-IPRT; hpt, bacterial gene for HPRT; POPOP, 1,4-bis[2-(5-phenyloxazolyl)] benzene; PPO, 2,5-diphenyloxazole; PRPP, pbosphoribosylpyrophosphate; PRT, pbosphoribosyltransferase; SDS-PAGE, sodium dodeeyl sulphate-polyaeylamidegel electrophoresis.

(IMP) which is then used in further purine metabolism [4]. HPRT is thus a vital enzyme in the metabolism of P. falciparum and for this reason could be a useful target for drug design. A eDNA clone encoding the parasite enzyme has been isolated and sequenced [5]. The inferred amino acid sequence shows important homologies with the enzyme of the host. However, the two sequences do diverge extensively even within the postulated active site [6,7], again suggesting that structural differences between the host and parasite enzymes could be exploited in chemotherapy. HPRT has been partially purified from cultured asexual blood stages of P. falciparum, strain FCB [3]. The enzyme differs from that of the host in a number of respects, notably its substrate specificity and apparent Km values. The peak of enzyme activity obtained from parasite extracts suggested that it contained a single enzyme species able to use guanine and xanthine as substrates, in addition to hypoxanthine. The study reported here provides strong confirmation of this conclusion. We report here high-level expression of the P. falciparum HPRT gene in bacteria. Parasite

0166-6851/90/$03.50 (~ Elsevier Science Publishers B.V. (Biomedical Division)

282 enzyme was made in an active form in both Escherichia coli and Salmonella typhimurium. Indeed, the parasite gene was able to complement an hpt mutant of S. typhimurium. This Salmonella strain opens the way to simple screening of candidate antimalarial drugs directed against the parasite HPRT. We discuss ways in which these recombinant bacteria can contribute to understanding of the parasite enzyme. Materials and Methods

Construction of plasmld pPfPRT2

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Construction of the recombinant plasmid pPIPR T2. The entire P. falciparum H P R T coding sequence was previously cloned from a pUC8 recombinant, 7-1a [5] into the HindlII and EcoRI site of a plasmid expression vector, pMS1S called pPfPRT1. Details concerning the construction of pPfPRT1 will be published elsewhere. The HindIII site in this plasmid is located one base upstream from the hprt initiation codon, A T G , (see Fig. 1) and EcoRI is 11 bases downstream from the stop codon, T A A . Fig. 1 shows how the open reading frame from A T G to T A A was recovered and inserted into the NdeI and EcoRI

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Bacterial strains and growth media. E. coli K12 strain NM654, a derivative of C600 [10], which is deleted in the hsd genes (N. Murray, personal communication) was used for the expression studies. S. typhimurium LT2 strain KP1684, purE1522 deoD203 hpt gpt, kindly donated by K. Jensen [9], was used for studying PRT activities and complementation. Plasmids propagated in E. coli were passaged in S. typhimurium LB5000 metA metE trpE leu hSdLT R - M + hsdsA R - M + hSdsB R - M + [11], which modifies but does not restrict foreign D N A , before transformation into the hsdR+M + strain, KP 1684 [9]. Luria broth [12] supplemented, where necessary, with ampiciUin (50 izg m1-1) was used for all liquid cultures. For complementation studies, minimal medium as described by Houlberg and Jensen [9] was used, supplemented with 1% agar (Bacto) and with amino acids (50 ~g ml-1), casamino acids (10 mg ml-1), nucleotide bases (15 I~g m1-1) and nucleosides (30 ~g m1-1) as stated in the text. The cultures were incubated at 30°C unless otherwise stated.

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Fig. 1. Construction of plasmid pPfPRT2. The high-level expressionvector, pJLAS03,is shownon the left. It carries the h repressor gene, cI, with the temperature-sensitivemutation 857, two k promoters in tandem, PL and PR, under the control of the repressor, and a translationinitiationregion (TIR) from the gene, atpE, encodingsubunit c of ATP synthase from E. coll. ForeignDNA is clonedinto a short polylinkercontaining NdeI and EcoRI sites. Transcriptionof the insert is repressed at 30"C and switched on at 42°C. The insert, containingthe hprt eDNA of P. falciparum was excised from pPfPRT1 (M. Shahabuddin, unpublished). The recombinant was recovered by transformation of E. coil, selecting ampiciUin-resistant clones (see Materialsand Methods). sites of the expression plasmid pJLA503 [8]. In brief, the plasmid pPfPRT1 was digested at the unique HindlII site preceding the 5' end of the A T G of the H P R T coding sequence and filled with D N A polymerase I (Klenow fragment). The blunt-ended linear plasmid was then digested at the EcoRI site located after the stop codon. The recovered cDNA insert was then gel-purified and ethanol-precipitated. The plasmid vector pJLA503 was cut at the unique NdeI site and

283 blunt ended with Klenow fragment as above. The NdeI-EcoRI polylinker fragment was then removed by digestion with EcoRI and ethanol precipitation. The purified eDNA was then ligated with the linear plasmid vector and transformed [13] into the E. coli NM654. Ampicillin resistant colonies were picked and screened by colony hybridisation [13] with PfHPRT eDNA, 7la [5]. One such positive strain was named SH2 and the recombinant plasmid was called pPfPRT2. The plasmid was purified and the structure and orientation of its insert were confirmed by restriction mapping. The recombinant plasmid was transformed into and purified from Salmonella typhimurium strain LB5000 (see above). This modified plasmid was then transformed into the S. typhimurium hpt mutant KP1684. One such transformant is called the SH4.

Induction of phosphoribosyl transferase and detection on SDS-PAGE. To study expression of the PfHPRT, a 5 ml culture of the bacterium was grown overnight (30~C) in Luria broth containing 50 p,g m1-1 ampicillin in a shaking water bath. To induce expression the temperature was shifted to 42°C for one hour. A 1.5-ml aliquot was centrifuged for one minute and the pelleted cells were resuspended in 50 p,1 of distilled water. To prepare the cells for electrophoresis, 50 p,1 of Laemmli sample buffer [14] were added, mixed and left at room temperature for 5 rain. The samples were then heated in a boiling water bath for 5 min and centrifuged for 10 min prior to use. 20 p,l of the supernatant was loaded on 12% polyacrylamide gel and electrophoresed at 50 mA constant current. The gels were stained with Coomassie Blue R250 [14]. For enzyme assays, 100 ml cultures were induced as above and the bacteria were collected by eentrifugation at 10000 x g for 10 min in a refrigerated centrifuge. They were washed once with the lysis buffer (225 mM Tris-HC1 buffer, pH 7.5, 5 mM MgSO4), resuspended in 10 ml of the same buffer and sonicated. The sonicate was centrifuged (45000 × g; 30 min) and the clear supernatant was used for enzyme assays.

Assay of PRT activities. Assay mixtures, in a final

volume of 50 Ixl, contained 225 mM Tris-HCl, pH 7.5, 5 mM MgSO4, 0.1% BSA, 2 mM PRPP and 100 p,M of [3H]hypoxanthine (Amersham) or [14C]guanine or [14C]xanthine (NEN, Dupont). The reactions were started by adding enzyme extract. The control contained no extract. The reactions were allowed to continue for 10 min at 37°C and stopped by heating at 100°C for 5 min. Precipitated proteins were pelleted by centrifugation for 10 min. Aliquots of 20 p,l of the reaction mixture were applied to Whatman No. 3 MM paper, previously spotted with a solution containing 0.1 p,mol of non radioactive mixture of the purine and its corresponding ribonucleotide. Chromatograms were developed in an ascending direction at room temperature for 2 h in 5% Na2HPO4:isoamyl alcohol, 2:1 (v/v). The extent of the conversion of the purines to corresponding ribonucleotides was determined by cutting out the ultraviolet light absorbing spots containing the marker ribonucleotide. The radioactivity of each spot was counted (Beckmann, LS7000, Liquid Scintillation System) in 2 ml Scintillation fluid (4 g PPO/0.01 g POPOP/50 ml Triton X-100 in a total volume of 150 ml of toluene). Results

Construction of pPfPRT2. The hprt gene of P. falciparum contains an intron [15] and is thus unsuitable for expression in prokaryotic cells. Instead we used a eDNA molecule which contains the entire hprt coding sequence [5]. It was inserted into a high level plasmid expression vector, pJLA503 (Fig. 1). Briefly, this vector [8] has in tandem two efficient promoters of phage h, PL and PR, an efficient translation initiation region and a multiple cloning site. Expression of insert DNA is controlled by the temperature-sensitive h repressor encoded by the ci857 gene carded on the plasmid. The hprt coding sequence was inserted between NdeI and EcoRI in the multiple cloning site and the recombinant plasmid, named pPt~RT2, was recovered and maintained in E. coli NM654 (see Materials and Methods).

Expression of P. falciparum hprt gene in E. coli. We sought expression of the parasite gene in above bacterium, named SH2 (see Materials and

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Fig. 3. Hypoxanthine phosphoribosyl transferase activities in recombinant and parent E. coli. Activities in bacterial extracts were measured by following incorporation [3H]hypoxanthine into IMP (see Materials and Methods). Data are expressed as percentages of the induced recombinant, SH2, (bar 3), which typically incorporated 60001) cpm in 10 rain. The parent, NM654, (bar 1) has 1% activity of the induced recombinant. The uninduced recombinant (bar 2) has 11% of the control (see text).

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Methods). Broth cultures grown at 30°C, with the X repressor active, were switched to 42°C for an hour, to induce expression of the parasite hprt gene, and analysed by SDS-PAGE. They synthesised a unique protein stained clearly by Coomassie Blue (Fig. 2, lane C). The uninduced culture (lane B) lacked the band, as did the parent bacterial strain, grown at 42°C (lane A). A control culture, containing the vector alone, also lacked the protein after induction (data not shown). The new protein has a relative molecular mass of 26 kDa, close to that predicted for the parasite enzyme by the coding sequence [5]. Moreover, after induction, the strain SH2, containing the recombinant plasmid, now has a very high level of HPRT activity, compared with the uninduced culture, and the parent, NM654, after induction (Fig. 3). We conclude that the P. falciparum hprt gene is expressed and that its product is active in E. coli. The elevated level of HPRT activity found even in the uninduced culture of SH2 may be due to a basal level of expression of the cloned gene.

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Expression of P. falciparum hprt in S. typhimurium. If the P. falciparum H P R T is active in bacteria, it may be possible to complement a bacterial mutant lacking the enzyme (hpt) with the parasite gene. Such a construct would be of great

285

value for investigating enzyme function and screening drugs. We chose to pursue this study in S. typhimuriurn, since an appropriate mutant of this organism was available [9]. The strain, KP1684, cannot synthesise purines de novo thanks to a mutation in pure (Fig. 4, step 1), it also has a deoD mutation (step 2) which blocks conversion of adenosine, inosine and guanosine to their cognate bases. In S. typhimurium two phosphoribosyl transferases are known, one encoded by gpt (step 3) uses xanthine or guanine as substrate, the other encoded by hpt (step 4) uses hypoxanthine [9]. In KP1684 both of these genes are inactive. This strain requires adenine and guanosine for growth (see Fig. 4). Its purine needs cannot be satisfied by hypoxanthine. The plasmid pPfPRT2 was transferred into KP1684 (see Materials and Methods). The recombinant strain, SH4, in expression studies carried out as before, showed a novel protein of 26 kDa after induction at 42°C (data not shown). Moreover, the H P R T activity in the induced extract was high, whereas the enzyme was undetected in the parent strain (Table I). Thus, the Salmonella recombinant, like that of E. coli, makes active

enzyme from the parasite gene. It was then tested for its ability to grow on minimal medium supplemented with hypoxanthine. We expected that SH4 would be able to grow at 42°C, if the parasite HPRT could complement the hpt mutation of the bacterium. However, no growth was observed. It turns out that the inability of these bacteria to grow on minimal medium with hypoxanthine is not due to a failure of the parasite hprt gene to complement. HPRT uses large amounts of its second substrate, PRPP, which is also needed for synthesis of pyrimidines, histidine and tryptophan [16]. Thus, a rapid increase in the level of active HPRT and of hypoxanthine itself could severely deplete the PRPP pool and consequently block other vital pathways. A depletion of the PRPP pool after addition of hypoxanthine to wild-type S. typhimurium has been reported [16]. This explanation was confirmed by plating the bacteria on minimal medium supplemented with hypoxan-

TABLE I Properties of P. falciparum hypoxanthine phosphoribosyl transferase expressed in S. typhimurium Enzyme source

Purine substrate Estimated specific activity (nmol (10 rain -1 mg -1)

KP1684

Hypoxanthine

ND

KP1684/ pJLAS03

Hypoxanthine

ND

SH4

Hypoxanthine Xanthine Guanine

60.8 (-+ 4.6) 5.79 (--- 1.2) 15.8 (-+ 1.9)

% Activity in presence of 1 mM 6-thioguanine

76 2.5 75

Under our conditions, the parent strain, KP1684, has no detectable PRT activity (Materials and Methods). The table also shows the estimated specific activities with hypoxanthine, xanthine and guanine (see Fig. 6). In each assay 1 Izl of extract was used. Specific activities shown are the average of at least three determinations. Standard deviations appear in brackets. Inhibition with 6-thioguanine was measured by adding the analogue to appropriate reaction mixtures (final concentration

1 raM).

Fig. 5. Complementation of an hpt mutation of S. typhimurium by P. falciparum hprt. A single colony of SH4 was inoculated into Luria broth and grown overnight (30°C), diluted (10 -2) into minimal medium with casaminoacids and cytidine (Materials and Methods) without hypoxanthine and spread on the selective plates which contained casaminoacids and cytidine and hypoxanthine where appropriate. (A) With hypoxanthine, 42°C; (B) no hypoxanthine, 42°C; (C) with hypoxanthine, 30°C; (D) no hypoxanthine, 30°C. Photographed after 24 h incubation.

286

thine, cytidine, histidine and tryptophan. They grew, albeit slowly, at 42°C on this medium whereas no growth was seen at 42° on the same medium lacking hypoxanthine or after incubation at 30°C (data not shown). Much faster growth and the same discrimination is seen on minimal medium with casamino acids, hypoxanthine and cytidine (Fig. 5). After 24 h at 42°C, colonies of SH4 reached 3 mm diameter (Fig. 5A), whereas no growth was observed on the plates incubated at 30°C with (Fig: 5C) or without (Fig. 5D) hypoxanthine. As expected, the bacteria could not grow at 42°C without hypoxanthine (Fig. 5B). We conclude that S. typhimurium is able to rely on the parasite gene to provide the HPRT needed to fulfill its purine requirements.

Studies on the substrate specificity of P. falciparum hypoxanthine phosphoribosyl transferase synthesised in S. typhimurium. When HPRT was purified from cultured parasites [3], the active fraction was found to phosphoribosylate both xanthine and guanine, in addition to hypoxanthine. These activities copurified at each step in the fractionation, suggesting that, unlike the host, P. falciparum has a single enzyme which can use all of these substrates. We have been able to test this proposal. Our S. typhimurium strain, SH4 hpt gpt pPfPRT, lacks the bacterial HPRT and GPRT, but has a single gene encoding the P. falciparum

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Fig. 6. Substrate specificity of P. falciparum hypoxanthine phosphoribosyl transferase expressed in S. typhimurium. Dependence of the incorporation of radiolabelled purines into corresponding ribomonophosphates on the volume of induced SH4 extracts (Materials and Methods). The lesser incorporation by higher volumes may be due to the presence of an increasing amount of contaminating enzymes in crude extract.

HPRT. If the hypothesis is correct, extracts of this strain, containing the single parasite enzyme, should be able to utilise both guanine and xanthine. The results in Fig. 6 confirm this conclusion (see also Table I). The extract of SH4 is able to phosphoribosylate all three bases. Moreover, the specific activities vary widely, hypoxanthine and guanine being preferred to xanthine. The enzyme extracted from the parasite has the same substrate preference [3]. We conclude that the parasite enzyme does have the property of being able to utilise all three bases. We now tested the effect of the competitive inhibitor, 6-thioguanine [17], on utilisation of the three substrates (Table I). At the concentration of the analogue chosen (1 mM), phosphoribosylation of hypoxanthine and guanine were inhibited by 25%. By contrast, the same amount of 6-thioguanine blocked more than 95% of activity for xanthine. A competitive inhibitor would be expected to affect xanthine utilisation more than that of the other bases if, as Queen et al. reported [3], the enzyme has a gm for this substrate (23 ixM) almost a hundred times greater than those for hypoxanthine (0.46 vLM) and guanine (0.30 v.M). Discussion

We show here that the HPRT of P. falciparum is made in a functional form by both E. coli and S. typhimurium from its cloned cDNA sequence. Indeed, at least the latter bacterium can use the parasite enzyme exclusively to satisfy its purine needs. This opens the possibility of a full functional analysis of the enzyme in a more tractable organism than the parent parasite. Comparisons of the amino acid sequences of phosphoribosyl transferases from a wide range of organisms have pointed to a 20-residue sequence thought to contain the catalytic domain [6,7]. This sequence is conserved in the P. falciparum hprt gene [5]. Since we are now able to synthesise the protein in large amounts, it may be possible to initiate structural studies on the parasite enzyme to test this model. In addition, the ease with which the parasite gene can now be manipulated will make it possible to combine site-directed mutation and functional studies to analyse the

287 structure of the active site of this enzyme. The P. falciparum enzyme is of particular interest in this regard, since it has proved to have a wider substrate specificity than other enzymes studied to date in detail [6,7]. Structural information m a y also provide a f r a m e w o r k to design candidate drugs directed against this important parasite enzyme. O u r S. typhimurium construct, only able to grow on hypoxanthine if the parasite enzyme is functioning, could provide a cheap and simple growth test to screen the candidates. The great instability of the H P R T in cultured parasite extracts has so far prevented developm e n t of specific antibody probes to locate the enzyme in the parasite. It should now be possible to m a k e antibodies to establish whether this enzyme is m a d e at all stages of the life-cycle and to discover whether the enzyme is free in the cytoplasm or associated with definite structures in the parasite. W h y should P f H P R T be able to use xanthine? The role of this enzyme in the mosquito m a y bear upon this question. In man, Plasmodium, like other parasites, predominantly uses the most available purine, hypoxanthine [4]. By contrast, mosquitoes, like other terrestrial insects, use purine metabolism in the excretion of nitrogen [18]. A m m o n i a , the end product of protein and amino acid catabolism, is condensed with glycine, f o r m a t e and aspartate to form purine ribotides. These are metabolised to xanthine which is later converted to .uric acid by xanthine oxidase [18]. This m a y result in a high concentration of xanthine in the hemocoel. In the mosquito, the parasite multiplies extensively [19], needing much m o r e purine than in man. Perhaps the parasite has evolved an H P R T able to exploit the available xanthine in the insect.

Acknowledgements W e t h a n k Angus King and David Melton for the generous gift of the P. falciparum hprt c D N A clone and support and advice. W e thank Michael G o m a n , R o b e r t Ridley and Chris Delves for e n c o u r a g e m e n t and discussions, D r K.F. Jensen and D r D e r e k Jamieson for S, typhimurium mutants, Professor N o r e e n Murray for E. coli

strains, Annie Wilson for preparing the manuscript and G r a h a m Brown for photography. D r Dick D ' A r i (Institut Jacques Monod, Paris) enlightened us about the importance of the P R P P pool in bacterial metabolism. M.S. holds a Scholarship awarded by the Association of C o m m o n wealth Universities. This work was supported by the M R C .

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Vol. 1., pp. 447-448. American Society for Microbiology, Washington DC. 17 Schimandle, M.C., Mole, L.A. and Sherman, I.W. (1987) Purification of hypoxanthineguanine phosphoribosyltransferase of Plasmodium lophurae. Mol. Biochem. Parasitol. 23, 39-45. 18 Gilmour, D. (1965) The Metabolism of Insects. Oliver and Boyd, Edinburgh. 19 Pringle, G. (1966) A quantitative study of naturally acquired malaria infections in Anopheles gambiae and Anopheles funestus. Trans. R. Soc. Trop. Med. Hyg. 60, 626-632.