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The in vitro uptake of tritiated nucleic acid precursors by Babesia spp. of cattle and mice
Inrernafionnl Journlrl for Parasirology.1978. Vol. 8. pp. 19-24. Pergamon Press. Printed in Great Britain.
THE IN VITRO UPTAKE OF TRITIATED NUCLEIC ACID PRECURSORS BY BABESZA SPP. OF CATTLE AND MICE A. D. ARC
IRVIN, E. R. YOUNG
and R. E.
PURNELL
Institute for Research on Animal Diseases, Compton, Newbury, Berkshire, U.K. (Received 22 April 1977)
Abstract-IRVIN A. D., YOUKG E. R. and PURNELLR. E. 1978. The in vitro uptake of tritiated nucleic acid precursors by Babesia spp. of cattle and mice. international Journal for Parasitology 8: 19-24. Blood and mice infected with Babesia microti and B. rodhaini, and from cattle infected with B. divergens and B. major, was incubated in Eagles medium for 24 h in the presence of tritiated purines and pyrimidines. Uptake of these compounds was assessed by liquid scintillation counting and by autoradiography. Hypoxanthine, adenosine and adenine were readily incorporated by all four species of parasites. Thymine, thymidine and uridine were generally not incorporated. Uptake of [aH]hypoxanthine by B. microti occurred within minutes of exposure to the precursor. The amount of [SH]hypoxanthine incorporated by B. rod/&&infected erythrocytes was proportional to the percentage of parasitized cells The results suggest that structural analogues of hypoxanthine and other purines may be incorporated and act against intra-erythrocytic Babesiu.
INDEX KEY WORDS: Babesia rnicroti: Babesia major; Bubesia divergens; Babesia rodhaini; tritium; thymine; thymidine; uridine; adenine; adenosine; hypoxanthine; purine; pyrimidine; nucleic acid ; autoradiography ; liquid scintillation counting; mice; cattle; erythrocyte.
INTRODUCTION STUDIES on the incorporation of radioactive purines and pyrimidines by different species of intracellular parasitic protozoa, have provided insight into the mechanisms of nucleic acid synthesis in these parasites. Most of such work has been conducted with malaria parasites, including Plasmodium berghei (Bungener & Nielsen, 1967, 1968; Van Dyke, Tremblay, Lantz & Szustkiewicz, 1970; Van Dyke, 1975), P. vinckei (Bungener & Nielsen, 1967, 1968), P. knowlesi (Polet & Barr, 1968; Gutteridge & Trigg, 1970), P. chabaudi (Schmidt, Walter & Konigk, 1974; Walter & Konigk, 1974) and P. fophurae (Walsh & Sherman, 1968). Similar work has been carried out with Coccidia (Ouellette, Strout & McDougaId, 1973; Morgan & Canning, 1974), Toxophzsma (Perrotto, Keister & Gelderman, 1971) and Theileria (Irvin & Stagg, 1977). We have applied the techniques of these workers to a study of the uptake of tritiated nucleic acid precursors by the intra-erythrocytic stages of Babesiu parasites of mice and cattle.
MATERIALS
AND METHODS
Parasites. Bubesiu rnicroti (Kings strain) rodhaini (Cox & Young, 1969) were originally
and B. supplied to us from King’s College, London; both parasites have been continuously maintained by blood passage in LAC/G strain mice. B. divergens (Eire 2 strain-Purnell, Brocklesby, Kitchenham & Young, 1976) and B. major
(Essex strain-Morzaria, Brocklesby, Harradine & Barnett, 1974) were obtained from splenectomised cattle experimentally infected at this Institute by inoculation of infected blood. Blood was collected from infected and uninfected animals into Anticlot (Clinton Laboratories, Santa Monica, CA, U.S.A.) by cardiac puncture (mice) or jugular venipuncture (cattle). After collection, blood samples were washed three times in phosphate buffered saline (PBS) (uH 7.2) and finallv susnended in PBS before cell ‘counting with a Couher counter (FNGC) (Coulter Electronics, Harpenden, Hertfordshire, U.K.). The percentage of parasitized erythrocytes (parasitaemia) was determined from the count of 500 erythrocytes in a Giemsa-stained smear. Tritiated nucleic acid precursors. The following tritiated purines were used: the bases [2-SH]adenine (specific activity (S.A.) 20 Ci/mmol) and [G-*H]hypoxanthine (S.A. 2.0 Ci/mmol) and the nucleoside [G-*H]adenosine (S.A. 38 Ci/mmol). The pyrimidines used were: the base [6-3H]thymine (S.A. 5 Ci/mmol), and the nucleosides [6-3H]thymidine (S.A. 5 Ci/mmol) and [5-3H]uridine (S.A. 5 Ci/mmol). All compounds were obtained from the Radiochemical Centre, Amersham, Bucks. Their uptake by parasitized and non-infected erythrocytes was measured by liquid scintillation counting and on autoradiography. Culture ofblood. Samples of blood containing approx. 6 x lo* erythrocytes were suspended in 25 cme Falcon flasks (Falcon Plastics, Oxnard, CA, U.S.A.) in 10 ml of Eagles Minimal Essential Medium supplemented with 100 i.u./ml of benzyl penicillin, 100 ug/ml streptomycin sulphate, 25 u/ml mycostatin, 50 mr+HEPES buffer (pH 7.2) and 10% foetal calf serum. Cultures were maintained at 37°C.
19
A. D.
20
IRVIN, E.
R.
YOUNG
Viability experiments. Preliminary experiments were conducted to confirm that parasites retained their viability following culture. B. r&rain&infected blood was cultured for 24 h, and then 1 x 10’ parasitized erythrocytes was inocurated into each of 7 mice. Smears of blood were prepared when cultures were set up and after 24 h. Liquid scintillation counting. The method used was based on that described by Gutteridge & Trigg (1970). In each experiment blood from infected and similar noninfected animals was processed simultaneously. Before incubation 100 pCi of tritiated purines or pyrimidines were added separately to each blood culture. Control flasks without tritiated purines or pyrimidines were also processed. After 24 h incubation, the erythrocytes from each flask were harvested after centrifugation at 1500 g and three washings in PBS. Cells were resuspended in 5 ml of PBS: 1 ml of each suspension was then transferred to 5 ml of 5’X trichloracetic acid at 4°C and duplicate 1 ml aliquots of this treated suspension were passed through 2.5 cm diameter 1.2 urn Millipore (Millipore, Park Royal, London, U.K.) filters. Each filter was washed twice with 5 ml of distilled water and then dried at 55°C. The dry filters were placed in scintillation vials containing 10 ml of scintillation fluid which contained 4.0 g PPO (2,5-Diphenyloxazole) -1~ 0.04 g dimethyl POPOP (I ,4-Di-2-4 methyl-5-phenyloxazolyl benzeneKoch Light) per litre of toluene, and counted in a Liquid Scintillation Analyser (Intertechnique, Versailles, France) at 8°C. Each sample was counted for 10 min; counts per minute were recorded, corrected to eliminate background and the mean of the two counts calculated. In order to compensate for different specific activities of the tritiated precursors, this figure was adjusted to assume a constant specific activity of 5 Ci/mmol. Tritiated precursors were incorporated by parasitized and non-parasitized blood and in order to obtain a figure which reflected the relative uptake of precursor by parasites alone the following formula was used (except Table 1) to express counts:
I-C c where I : count per minute for infected blood; C = count per minute for non-infected blood. In addition to studying the 24-h uptake of tritiated precursors by four species of Babesio, two other experiments were performed. In the first 19. nricroti-infected blood was pooled from 10 mice, parasitaemia was determined and blood was cultured as before in 20 x 5 ml samples of medium each containing 10 pCi of [SH]hypoxanthine. Pairs of samples were harvested on IO occasions after 1, 5, 10 and 30 min and I, 2, 4, 6, 12 and 24 h incubation. The uptake of [3H]hypoxanthine was calculated as before. Paired controls of non-infected blood were treated and examined simultaneously. In the second experiment a group of mice was infected with B. rodhaini, blood smears were prepared daily and parasitaemias determined. Mice with different degrees of parasitaemia were killed and blood from them was processed as before and incubated for 24 h in the presence of 10 pCi of [SH]hypoxanthine/5 ml of medium. The uptake of [3H]hypoxanthine was determined for infected and noninfected control blood. Autoradiography. When harvesting blood for liquid scintillation counting, smears were also prepared on a number of occasions for autoradiography. Slides were
and R. E. PURNELL
I.J.P. VOL.8. 1978
fixed in methanol, extracted for 30 min in 5% trichloracetic acid at 4°C then processed as previously described (Irvin, Brown, Boarer, Crawford & Kanhai, 1974). RESULTS Viability experimetzts
All mice died following inoculation with B. rhorlaini-infected blood that had been cultured for 24 h. The mean time to death was 6.3 days, as compared with 6.7 days in mice similarly inoculated with fresh blood. The parasitaemia of blood at the time of collection was 21.2”/, (500 cells counted) and after 24 h incubation was 24.2 T!. Parasites appeared morphologically normal. Uptake
qf’ tritiated precursors b.v diflkrettt Babesia Table 1 presents results obtained after incubation of blood from control and B. major infected cattle with tritiated nucleic acid precursors. A similar pattern was obtained with the other parasites in both mouse and bovine erythrocytes. In infected blood there was a marked uptake of purines, especially adenine and hypoxanthine, which was greatly in excess of the uptake by non-infected blood. There was very little uptake of pyrimidines by either infected or non-infected blood. Autoradiography showed that most of the pyrimidine uptake was accounted for by incorporation into white cells.
TABLE I-UPTAKE BOVINE
BLOOD
OF [3H]~~~~~~~ ACID PRECURSORS BY FROM CONTROL AND B. tT.'ajOY-INFECTED ANIMALS
[3H] labelled precursor Control blood Infected blood ~.~ _. __ _____.___ ~~. Pyrimidines 15.5 10.1 Thymine 45.0 Thymidine Il.1 21.8 62.8 Uridine Purines 8872.2 228.7 Adenine 73257 Adenosine 39.3 84973.4 361.2 Hypoxanthine Parasitaemia -= 23.2%. The figures are net counts per minute recorded after blood was incubated for 24 h in the presence of precursor.
Table 2 shows the uptake of precursors by parasites after the counts had been corrected to eliminate uptake of precursors by blood cells alone. Purines were incorporated at high levels by all four species of parasite, but pyrimidines were incorporated only by 3. rodhaini. Autoradiography confirmed the results of the liquid scintillation analysis. Intra-erythrocytic parasites were readily labelled by purines (Fig. 1) but extra-erythrocytic forms were usually unlabelled. Reticulocytes and leukocytes showed a low level of incorporation
of [‘HIpurines
and pyrimidines.
I.J.P. VOL. 8. 1978
Uptake of tritiated nucleic acid precursors by Babesia
21
TABLE ~-UPTAKE OF [aH]~~~~~~~ ACID PRECURSORS BY ERYTHROCYTES PARASITIZED WITH DIFFERENT SPECIES OF Babesia: ASSESSMEKT By LIQUID SCINTILLA~ON COUNTNG AFTER 24 h EXPOSURE TO PRECURSOR
[*HI labelled precursors Pyrimidines Thymine Thymidine Uridine Purines Adenine Adenosine Hypoxanthine Parasitaemia
(%)
B. microti
ND* 0.6 0.1
-0.9 24.2
B. major
B. divergens
10.5
-0.3 3.0 1.9
@l 2.4 2.0
3.5 ND 44.0
6.3 20.2 65.0
37.8 185.4 234.2
8.0 208.1 34.3
7.0
20.4
23.2
22.5
*ND = not done. The figures give the ratio of the incorporation
FIG. 1. Autoradiograph
B. rodhaini
by the parasites to that by uninfected blood.
of B. microti-infected blood, 24 h after incubation [3H]hypoxanthine.
in the presence
of
22
A. D. IRVIN,E. R. YOUNG and R.
Uptake difirent
of [3H]hypoxanthine time intervats
by B.
microti
after
Table 3 shows that there was a rapid and progressive increase in the uptake of [3H]hypoxanthine by B. microti infected erythrocytes up to 12 h. After 12 h further uptake occurred but the rate was reduced. The times given indicate when preparation of samples began, there was inevitably some further incorporation of [3H]hypoxanthine during handling, but the results nevertheless clearly show that there was significant incorporation of [3H]hypoxanthine after only a few minutes exposure. TABLE ~-UPTAKE OF [3H]~~~~~~~~I~~ BY B. microtiBLOOD (PARASITAEMIA 38%) AFTER DIFFERENT PERIODS OF INCUBATION
AVERTED Period
of incubation
Uptake
of [3H]hypoxanthine
1 min 5 min 10 min 30 min lh 2h 4h 6h 12 h 24 h
1.2 5.6 9.5 28.3 55.8 113.2 142.1 178.5 333.4 352.7
The figures give the ratio of the incorporation parasites to that by uninfected blood.
Uptake of [3H]hypoxanthine diflerent parasitaemias
by
by the
B. rodhaini
at
Table 4 shows that the amount of [3H]hypoxanthine incorporated after 24 h by B. rodhainiinfected erythrocytes was proportional to the parasitaemia. Significant incorporation was not detected until a parasitaemia of 5% was reached. At the highest parasitaemia there was some indication that the level of incorporation was falling. TABLE ~-UPTAKE OF [3H]~~~~~~~~~~~~ BY B. rodhainiINFECTED BLOOD WITH DIFFERENT DEGREES OF PARASITAEMIA, RECORDED AFTER 24 h INCUBATION Parasitaemia
%
Uptake
NPS*
of [3H]hypoxanthine
_
0.1 0.2 0.03 0.2 1.5 7.6 7.9 24.5 20.3
*NPS = infected but no parasites seen. The figures give the ratio of the incorporation parasites to that by uninfected blood.
by the
DISCUSSION
Babesia parasites maintained were morphologically normal
ill vitro over 24 h
and
retained
their
E. PURNELL
I.J.P.
VOL.
8.
1978
viability and infectivity. Maintenance of parasites by this method has now become standard in this laboratory, and has proved completely suitable for subsequent in vitro studies with Babesia (in preparation). The results of the current work clearly show that Babesia spp. of cattle and mice can incorporate free purines. These findings are similar to those of the malariologists mentioned above. Hypoxanthine appeared to be the purine most readily incorporated (Table 2), and for this reason was chosen for subsequent experiments (Tables 3 and 4). In a more detailed study with P. berghei, Van Dyke (1975) found that the level of incorporation was in the order hypoxanthine - adenosine > ;, adenine. A similar order of incorporation was noted in our work. [“HIPurines were incorporated into cells (mostly leucocytes and reticulocytes) of control blood, but the amount incorporated by these cells was trivial when compared with that incorporated by parasites. Consequently any change in reticulocyte or leukocyte numbers resultant from Babesia infection caused a negligible change in the ‘background’ count of infected blood. This meant that the count given by control blood could be used to provide the ‘background’ count for the infected blood. The counts for [“HIpurine uptake given in Table 2 and for [“HIhypoxanthine uptake in Tables 3 and 4 therefore represent uptake of these compounds essentially by parasites since ‘background’ has been largely eliminated. The rapid movement of purines across erythrocyte membranes is well documented (Mager, Hershko, Zeitlin-Beck, Shoshani & Razin, 1967; Berlin & Oliver, 1975) and is clearly confirmed by the resu!ts in Table 3, which show that significant levels of [3H]hypoxanthine were detected in parasitized erythrocytes within minutes of exposure. Furthermore, the amount of [3H]hypoxanthine incorporated bore a direct relationship to the parasitaemia (Table 4). The uptake of pyrimidines was more difficult to assess because of the relative inability of these compounds to permeate mature erythrocyte membranes (Lieu, Hudson, Brown, & White, 1971; Berlin & Oliver, 1975). The incorporation which did occur may have resulted from parasite-induced changes of the erythrocyte membrane increasing its permeability, such changes have been demonstrated to occur in B. rodhailli-infected erythrocytes (Homewood, Neame & Momen, 1975) and may well account for the high level of incorporation of [“Hlthymidine and [“Hluridine by B. rodhaini in the current work. Another reason for the high level of incorporation of pyrimidines by B. rocihaini-infected blood as compared with blood infected by the other parasites, may relate to the more rapid reticulocyte response induced by B. rodhaini (Dolan, 1974) and to the parasite’s possible preference for immature erythro-
1.1.p. VOL. 8. 1978
Uptake of Mated
nucleic acid precursors by Babesia
cytes (Nowell, 1969). In another experiment (Irvin & Young, unpublished) it was found that blood infected with Plusmodium yoelii, a rodent parasite with a predilection for immature erythrocytes, incorporated both purines and pyrimidines at high levels; whereas blood infected with P. chabaudi, which invades mature erythrocytes, incorporated only purines. The
present
work
demonstrates
parasites readily incorporate whether
that
Bubesiu
free purines in vitro,
or not they depend on these base compounds
for synthesising nucleic acids in viva remains to be determined. In many cell lines. if de nova synthesis of purines is blocked, nucleic acid synthesis can proceed via a salvage pathway utilizing exogenous hypoxanthine which is converted to inosinic acid by the enzyme hypoxanthine guanine phosphoribosyl transferase (EC 2.4.2.8) (Szybalska & Szybalski 1962). Similarly the intraerythrocytic stages of plasmodia are apparently unable to synthesize purine nucleotides de nova and depend on the uptake of preformed compounds (Bungener & Nielsen, 1968; Walsh & Sherman, 1968; Walter & Konigk, 1974). If the synthesis of nucleic acids by Babesia is also dependent on such a salvage pathway, as could be inferred by the very ready uptake of hypoxanthine, a possible site for chemical attack is offered. A starting point for such an attack would be the use of drugs such as 6-thioguanine, 8-azaguanine and 6-mercaptopurine, structural analogues of hypoxanthine, whose therapeutic properties have been well explored in the treatment of neoplasia (Connors, 197 1). Studies on the uptake of tritiated nucleic acid precursors by Plasmodium spp. have already been helpful in assessing the effects of various antimalarial compounds; our own studies and those of Chiodini (1973) indicate a similar application for the study of anti-babesial drugs.
CONNORST. A. 1971. Inhibition
precursors
are grateful to Dr. B. F. Sansom for his advice and Drs. N. McHardy and C. Ginger for helpful comments on the manuscript. We also thank Mr. C. Mallinson for carrying out scintillation counts, Mr. I. Jebbett for photography and Mr. D. Hendry for technical
assistance. REFERENCES BERLIN R. D. & OLIVER J. M. 1975. Membrane transport of purine and pyrimidine bases and nucleosides in animal cells. International Review of Cytology 42: 286-336. BUNGENER W. & NIELSON G. 1967. Nukleinsaurenstoffwechsel bei experimenteller malaria. I. Zeitschrifi fiir Tropenmedizin und Parasitoloaie 18: 456-462. BUNG~NER W. & NIELSON G. l&8. Nukleinsaurenstoffwechsel bei experimenteller malaria. II. Zeitschrift fB/ Tropenmedizin und Parasitologic 19: 185-197. CHIODINI P. L. 1973. In vifro culture of Babesiu. Transuctions of the Royal Society of Tropical Medicine and Hygiene 67: 27-28.
of nucleic
of the synthesis of the acids (DNA and RNA) In
Fundamentals of BiochemicalPharmacology (Edited by BACQ Z. M., CAPEK R., PAOLET~~ R. & RENSON J.). Pergamon Press, Oxford.
Cox F. E. G. & YOUNG A. S. 1969. Acquired immunity to Babesia microti and B. rodhaini in mice. Parasitology 59: 257-268. DOLAN T. T. 1974. Mechanisms of pathogenesis experrmental babesiosis. Ph.D. Thesis, Edinburgh.
in
GUTTERIDGE W. E. & TRIGG P. I. 1970. Incorporation of radioactive precursors into DNA and RNA of Plusmedium knonlesi in vitro. Journal of Protozoology 17: 89-96. HOMEWOOD C. A., NEAME K. D. & MOMEN H. 1975. Permeability of erythrocytes from mice infected with Babesia rodhaini. Annals of Tropical Medicine and Parasitology 69: 429-434. IRVIN A. D., BROWN C. G. D., BOARER C. D. H., CRAWFORD J. G. & KANHA~ G. K. 1974. Autoradio-
graphic evidence for the occurrence of cell fusion in cultures of Theileriu-infected bovine lymphoid cells. Research in Veterinary Science 16: 137-142. IRVIN A. D. & STAGG D. A. 1977. Theileria parva: purine and pyrimidine metabolism and the action of folate antagonists in parasitized bovine lymphoid cells. Experimental Parasitology 41: 172-l 85. LIEU T. S., HUDSON R. A., BROWN R. K. & WHITE B. C. 1971. Transport of pyrimidine nucleosides across human erythrocyte membranes. Biochimica et Biophysica Acta 241: 884-893. MAGER J., HERSHKO A., ZEITLIN-BECK R., SHOSHAN~ T. & RAZIN A. 1967. Turnover or purine nucleotides in rabbit erythrocytes. Biochimica et Biophysics Acta 149: 50-58. MORGAN K. & CANNING E. U. 1974. Incorporation of r3Hlthvmidine and VHladenosine bv Eimeria tenella grown-in chick embryos Journal oj Parasitology 60: 364-367. MORZAR~A S. P., BROCKLESBY D. W., HARRADINE D. L. & BARNETT S. F. 1974. Bubesia major in Britain: in-
fectivity Acknowledgements-We
23
of
suspensions
derived
Haemaphysalis punctata nymphs. for Parasitology 4 : 437-438.
from
ground-up
International
Journal
NOWELL F. 1969. The blood picture resulting from Nuttalia (Babesia) rodhaini and Nuttalia (Bubesiu) microti infections in rats and mice. Parasitology 59: 991-1004. OUELLETTE C. A., STROU~ R. G. & MCDOUCALD L. R. 1973. Incorporation of radioactive pyrimidine nucleosides into DNA and RNA of Eimeriu tenella (Coccidia) cultured in vitro. Journal of Protozoology 20: 150-153. PERROTTO J., KEISTER D. B. & GELDERMAN A. H. 1971. Incorporation of precursors into Toxoplasma DNA. Journal of Protozoology 18: 470-473. POLET H. & BARR C. F. 1968. DNA, RNA and protein synthesis in erythrocytic forms of PIasmodium knowlesi. American Journal of Tropical Medicine and Hygiene 17: 672-679. PURNELL R. E., BROCKLFSBY D. W., KITCHENHAM B. A. & YOUP~G E. R. 1976 A. statistical comparison of the behaviour of five British isolates of Babesia divergens in splenectomised calves. Journal of Comparative _.. _.___. Yatho!ogy 116: 609-614.
24
A. D. IRVIN,E. R. YOUNG and R. E. PURNELL
SCHMIDT G., WALTER R. D. & KONIGK E. 1974. Adenosine kinase from normal mouse erythrocytes and from Plasmodium chabaudi: partial purification and characterization. TropenmedizinundParaCtologie25: 301-308. SZYBALSKA E. & SZYBALSKI W. 1962. Genetics of human cell lines. IV. DNA-mediated heritable transformation of a biochemical trait. Proceedings of the National Academy of Science 48: 2026-2034. VAN DYKE K. 1975. Comparison of tritiated hypoxanthine, adenine and adenosine for purine-salvage incorporation into nucleic acids of the malarial parasite, Plasmodium berghei. Tropenmedizin und Parasitologic 26: 232-238.
1.J.P. VOL. 8. 1978
VAN DYKE K., TREMBLAY G. C., LANTZ C. H. & Szus-rKIEWICZ C. 1970. The source of purine and pyrimidines in Plasmodium berghei. American Journal of Tropical Medicine and Hygiene 19: 202-208. WALSH C. J. & SHERMAN I. W. 1968. Purine and pyrimidine synthesis by the avian malaria parasite Plasmodium lophurae. Journal of Protozoology 15: 185-197. WALTER R. D. & KONIGK E. 1974. Hypoxanthine guanine phosphoribosyl transferase and adenine phosphoribosyl transferase from Plasmodium chabaudi, purification and properties. Tropenmedizin und Parasitologic 25: 227-235.
THE IN VITRO UPTAKE OF TRITIATED NUCLEIC ACID PRECURSORS BY BABESZA SPP. OF CATTLE AND MICE A. D. ARC
IRVIN, E. R. YOUNG
and R. E.
PURNELL
Institute for Research on Animal Diseases, Compton, Newbury, Berkshire, U.K. (Received 22 April 1977)
Abstract-IRVIN A. D., YOUKG E. R. and PURNELLR. E. 1978. The in vitro uptake of tritiated nucleic acid precursors by Babesia spp. of cattle and mice. international Journal for Parasitology 8: 19-24. Blood and mice infected with Babesia microti and B. rodhaini, and from cattle infected with B. divergens and B. major, was incubated in Eagles medium for 24 h in the presence of tritiated purines and pyrimidines. Uptake of these compounds was assessed by liquid scintillation counting and by autoradiography. Hypoxanthine, adenosine and adenine were readily incorporated by all four species of parasites. Thymine, thymidine and uridine were generally not incorporated. Uptake of [aH]hypoxanthine by B. microti occurred within minutes of exposure to the precursor. The amount of [SH]hypoxanthine incorporated by B. rod/&&infected erythrocytes was proportional to the percentage of parasitized cells The results suggest that structural analogues of hypoxanthine and other purines may be incorporated and act against intra-erythrocytic Babesiu.
INDEX KEY WORDS: Babesia rnicroti: Babesia major; Bubesia divergens; Babesia rodhaini; tritium; thymine; thymidine; uridine; adenine; adenosine; hypoxanthine; purine; pyrimidine; nucleic acid ; autoradiography ; liquid scintillation counting; mice; cattle; erythrocyte.
INTRODUCTION STUDIES on the incorporation of radioactive purines and pyrimidines by different species of intracellular parasitic protozoa, have provided insight into the mechanisms of nucleic acid synthesis in these parasites. Most of such work has been conducted with malaria parasites, including Plasmodium berghei (Bungener & Nielsen, 1967, 1968; Van Dyke, Tremblay, Lantz & Szustkiewicz, 1970; Van Dyke, 1975), P. vinckei (Bungener & Nielsen, 1967, 1968), P. knowlesi (Polet & Barr, 1968; Gutteridge & Trigg, 1970), P. chabaudi (Schmidt, Walter & Konigk, 1974; Walter & Konigk, 1974) and P. fophurae (Walsh & Sherman, 1968). Similar work has been carried out with Coccidia (Ouellette, Strout & McDougaId, 1973; Morgan & Canning, 1974), Toxophzsma (Perrotto, Keister & Gelderman, 1971) and Theileria (Irvin & Stagg, 1977). We have applied the techniques of these workers to a study of the uptake of tritiated nucleic acid precursors by the intra-erythrocytic stages of Babesiu parasites of mice and cattle.
MATERIALS
AND METHODS
Parasites. Bubesiu rnicroti (Kings strain) rodhaini (Cox & Young, 1969) were originally
and B. supplied to us from King’s College, London; both parasites have been continuously maintained by blood passage in LAC/G strain mice. B. divergens (Eire 2 strain-Purnell, Brocklesby, Kitchenham & Young, 1976) and B. major
(Essex strain-Morzaria, Brocklesby, Harradine & Barnett, 1974) were obtained from splenectomised cattle experimentally infected at this Institute by inoculation of infected blood. Blood was collected from infected and uninfected animals into Anticlot (Clinton Laboratories, Santa Monica, CA, U.S.A.) by cardiac puncture (mice) or jugular venipuncture (cattle). After collection, blood samples were washed three times in phosphate buffered saline (PBS) (uH 7.2) and finallv susnended in PBS before cell ‘counting with a Couher counter (FNGC) (Coulter Electronics, Harpenden, Hertfordshire, U.K.). The percentage of parasitized erythrocytes (parasitaemia) was determined from the count of 500 erythrocytes in a Giemsa-stained smear. Tritiated nucleic acid precursors. The following tritiated purines were used: the bases [2-SH]adenine (specific activity (S.A.) 20 Ci/mmol) and [G-*H]hypoxanthine (S.A. 2.0 Ci/mmol) and the nucleoside [G-*H]adenosine (S.A. 38 Ci/mmol). The pyrimidines used were: the base [6-3H]thymine (S.A. 5 Ci/mmol), and the nucleosides [6-3H]thymidine (S.A. 5 Ci/mmol) and [5-3H]uridine (S.A. 5 Ci/mmol). All compounds were obtained from the Radiochemical Centre, Amersham, Bucks. Their uptake by parasitized and non-infected erythrocytes was measured by liquid scintillation counting and on autoradiography. Culture ofblood. Samples of blood containing approx. 6 x lo* erythrocytes were suspended in 25 cme Falcon flasks (Falcon Plastics, Oxnard, CA, U.S.A.) in 10 ml of Eagles Minimal Essential Medium supplemented with 100 i.u./ml of benzyl penicillin, 100 ug/ml streptomycin sulphate, 25 u/ml mycostatin, 50 mr+HEPES buffer (pH 7.2) and 10% foetal calf serum. Cultures were maintained at 37°C.
19
A. D.
20
IRVIN, E.
R.
YOUNG
Viability experiments. Preliminary experiments were conducted to confirm that parasites retained their viability following culture. B. r&rain&infected blood was cultured for 24 h, and then 1 x 10’ parasitized erythrocytes was inocurated into each of 7 mice. Smears of blood were prepared when cultures were set up and after 24 h. Liquid scintillation counting. The method used was based on that described by Gutteridge & Trigg (1970). In each experiment blood from infected and similar noninfected animals was processed simultaneously. Before incubation 100 pCi of tritiated purines or pyrimidines were added separately to each blood culture. Control flasks without tritiated purines or pyrimidines were also processed. After 24 h incubation, the erythrocytes from each flask were harvested after centrifugation at 1500 g and three washings in PBS. Cells were resuspended in 5 ml of PBS: 1 ml of each suspension was then transferred to 5 ml of 5’X trichloracetic acid at 4°C and duplicate 1 ml aliquots of this treated suspension were passed through 2.5 cm diameter 1.2 urn Millipore (Millipore, Park Royal, London, U.K.) filters. Each filter was washed twice with 5 ml of distilled water and then dried at 55°C. The dry filters were placed in scintillation vials containing 10 ml of scintillation fluid which contained 4.0 g PPO (2,5-Diphenyloxazole) -1~ 0.04 g dimethyl POPOP (I ,4-Di-2-4 methyl-5-phenyloxazolyl benzeneKoch Light) per litre of toluene, and counted in a Liquid Scintillation Analyser (Intertechnique, Versailles, France) at 8°C. Each sample was counted for 10 min; counts per minute were recorded, corrected to eliminate background and the mean of the two counts calculated. In order to compensate for different specific activities of the tritiated precursors, this figure was adjusted to assume a constant specific activity of 5 Ci/mmol. Tritiated precursors were incorporated by parasitized and non-parasitized blood and in order to obtain a figure which reflected the relative uptake of precursor by parasites alone the following formula was used (except Table 1) to express counts:
I-C c where I : count per minute for infected blood; C = count per minute for non-infected blood. In addition to studying the 24-h uptake of tritiated precursors by four species of Babesio, two other experiments were performed. In the first 19. nricroti-infected blood was pooled from 10 mice, parasitaemia was determined and blood was cultured as before in 20 x 5 ml samples of medium each containing 10 pCi of [SH]hypoxanthine. Pairs of samples were harvested on IO occasions after 1, 5, 10 and 30 min and I, 2, 4, 6, 12 and 24 h incubation. The uptake of [3H]hypoxanthine was calculated as before. Paired controls of non-infected blood were treated and examined simultaneously. In the second experiment a group of mice was infected with B. rodhaini, blood smears were prepared daily and parasitaemias determined. Mice with different degrees of parasitaemia were killed and blood from them was processed as before and incubated for 24 h in the presence of 10 pCi of [SH]hypoxanthine/5 ml of medium. The uptake of [3H]hypoxanthine was determined for infected and noninfected control blood. Autoradiography. When harvesting blood for liquid scintillation counting, smears were also prepared on a number of occasions for autoradiography. Slides were
and R. E. PURNELL
I.J.P. VOL.8. 1978
fixed in methanol, extracted for 30 min in 5% trichloracetic acid at 4°C then processed as previously described (Irvin, Brown, Boarer, Crawford & Kanhai, 1974). RESULTS Viability experimetzts
All mice died following inoculation with B. rhorlaini-infected blood that had been cultured for 24 h. The mean time to death was 6.3 days, as compared with 6.7 days in mice similarly inoculated with fresh blood. The parasitaemia of blood at the time of collection was 21.2”/, (500 cells counted) and after 24 h incubation was 24.2 T!. Parasites appeared morphologically normal. Uptake
qf’ tritiated precursors b.v diflkrettt Babesia Table 1 presents results obtained after incubation of blood from control and B. major infected cattle with tritiated nucleic acid precursors. A similar pattern was obtained with the other parasites in both mouse and bovine erythrocytes. In infected blood there was a marked uptake of purines, especially adenine and hypoxanthine, which was greatly in excess of the uptake by non-infected blood. There was very little uptake of pyrimidines by either infected or non-infected blood. Autoradiography showed that most of the pyrimidine uptake was accounted for by incorporation into white cells.
TABLE I-UPTAKE BOVINE
BLOOD
OF [3H]~~~~~~~ ACID PRECURSORS BY FROM CONTROL AND B. tT.'ajOY-INFECTED ANIMALS
[3H] labelled precursor Control blood Infected blood ~.~ _. __ _____.___ ~~. Pyrimidines 15.5 10.1 Thymine 45.0 Thymidine Il.1 21.8 62.8 Uridine Purines 8872.2 228.7 Adenine 73257 Adenosine 39.3 84973.4 361.2 Hypoxanthine Parasitaemia -= 23.2%. The figures are net counts per minute recorded after blood was incubated for 24 h in the presence of precursor.
Table 2 shows the uptake of precursors by parasites after the counts had been corrected to eliminate uptake of precursors by blood cells alone. Purines were incorporated at high levels by all four species of parasite, but pyrimidines were incorporated only by 3. rodhaini. Autoradiography confirmed the results of the liquid scintillation analysis. Intra-erythrocytic parasites were readily labelled by purines (Fig. 1) but extra-erythrocytic forms were usually unlabelled. Reticulocytes and leukocytes showed a low level of incorporation
of [‘HIpurines
and pyrimidines.
I.J.P. VOL. 8. 1978
Uptake of tritiated nucleic acid precursors by Babesia
21
TABLE ~-UPTAKE OF [aH]~~~~~~~ ACID PRECURSORS BY ERYTHROCYTES PARASITIZED WITH DIFFERENT SPECIES OF Babesia: ASSESSMEKT By LIQUID SCINTILLA~ON COUNTNG AFTER 24 h EXPOSURE TO PRECURSOR
[*HI labelled precursors Pyrimidines Thymine Thymidine Uridine Purines Adenine Adenosine Hypoxanthine Parasitaemia
(%)
B. microti
ND* 0.6 0.1
-0.9 24.2
B. major
B. divergens
10.5
-0.3 3.0 1.9
@l 2.4 2.0
3.5 ND 44.0
6.3 20.2 65.0
37.8 185.4 234.2
8.0 208.1 34.3
7.0
20.4
23.2
22.5
*ND = not done. The figures give the ratio of the incorporation
FIG. 1. Autoradiograph
B. rodhaini
by the parasites to that by uninfected blood.
of B. microti-infected blood, 24 h after incubation [3H]hypoxanthine.
in the presence
of
22
A. D. IRVIN,E. R. YOUNG and R.
Uptake difirent
of [3H]hypoxanthine time intervats
by B.
microti
after
Table 3 shows that there was a rapid and progressive increase in the uptake of [3H]hypoxanthine by B. microti infected erythrocytes up to 12 h. After 12 h further uptake occurred but the rate was reduced. The times given indicate when preparation of samples began, there was inevitably some further incorporation of [3H]hypoxanthine during handling, but the results nevertheless clearly show that there was significant incorporation of [3H]hypoxanthine after only a few minutes exposure. TABLE ~-UPTAKE OF [3H]~~~~~~~~I~~ BY B. microtiBLOOD (PARASITAEMIA 38%) AFTER DIFFERENT PERIODS OF INCUBATION
AVERTED Period
of incubation
Uptake
of [3H]hypoxanthine
1 min 5 min 10 min 30 min lh 2h 4h 6h 12 h 24 h
1.2 5.6 9.5 28.3 55.8 113.2 142.1 178.5 333.4 352.7
The figures give the ratio of the incorporation parasites to that by uninfected blood.
Uptake of [3H]hypoxanthine diflerent parasitaemias
by
by the
B. rodhaini
at
Table 4 shows that the amount of [3H]hypoxanthine incorporated after 24 h by B. rodhainiinfected erythrocytes was proportional to the parasitaemia. Significant incorporation was not detected until a parasitaemia of 5% was reached. At the highest parasitaemia there was some indication that the level of incorporation was falling. TABLE ~-UPTAKE OF [3H]~~~~~~~~~~~~ BY B. rodhainiINFECTED BLOOD WITH DIFFERENT DEGREES OF PARASITAEMIA, RECORDED AFTER 24 h INCUBATION Parasitaemia
%
Uptake
NPS*
of [3H]hypoxanthine
_
0.1 0.2 0.03 0.2 1.5 7.6 7.9 24.5 20.3
*NPS = infected but no parasites seen. The figures give the ratio of the incorporation parasites to that by uninfected blood.
by the
DISCUSSION
Babesia parasites maintained were morphologically normal
ill vitro over 24 h
and
retained
their
E. PURNELL
I.J.P.
VOL.
8.
1978
viability and infectivity. Maintenance of parasites by this method has now become standard in this laboratory, and has proved completely suitable for subsequent in vitro studies with Babesia (in preparation). The results of the current work clearly show that Babesia spp. of cattle and mice can incorporate free purines. These findings are similar to those of the malariologists mentioned above. Hypoxanthine appeared to be the purine most readily incorporated (Table 2), and for this reason was chosen for subsequent experiments (Tables 3 and 4). In a more detailed study with P. berghei, Van Dyke (1975) found that the level of incorporation was in the order hypoxanthine - adenosine > ;, adenine. A similar order of incorporation was noted in our work. [“HIPurines were incorporated into cells (mostly leucocytes and reticulocytes) of control blood, but the amount incorporated by these cells was trivial when compared with that incorporated by parasites. Consequently any change in reticulocyte or leukocyte numbers resultant from Babesia infection caused a negligible change in the ‘background’ count of infected blood. This meant that the count given by control blood could be used to provide the ‘background’ count for the infected blood. The counts for [“HIpurine uptake given in Table 2 and for [“HIhypoxanthine uptake in Tables 3 and 4 therefore represent uptake of these compounds essentially by parasites since ‘background’ has been largely eliminated. The rapid movement of purines across erythrocyte membranes is well documented (Mager, Hershko, Zeitlin-Beck, Shoshani & Razin, 1967; Berlin & Oliver, 1975) and is clearly confirmed by the resu!ts in Table 3, which show that significant levels of [3H]hypoxanthine were detected in parasitized erythrocytes within minutes of exposure. Furthermore, the amount of [3H]hypoxanthine incorporated bore a direct relationship to the parasitaemia (Table 4). The uptake of pyrimidines was more difficult to assess because of the relative inability of these compounds to permeate mature erythrocyte membranes (Lieu, Hudson, Brown, & White, 1971; Berlin & Oliver, 1975). The incorporation which did occur may have resulted from parasite-induced changes of the erythrocyte membrane increasing its permeability, such changes have been demonstrated to occur in B. rodhailli-infected erythrocytes (Homewood, Neame & Momen, 1975) and may well account for the high level of incorporation of [“Hlthymidine and [“Hluridine by B. rodhaini in the current work. Another reason for the high level of incorporation of pyrimidines by B. rocihaini-infected blood as compared with blood infected by the other parasites, may relate to the more rapid reticulocyte response induced by B. rodhaini (Dolan, 1974) and to the parasite’s possible preference for immature erythro-
1.1.p. VOL. 8. 1978
Uptake of Mated
nucleic acid precursors by Babesia
cytes (Nowell, 1969). In another experiment (Irvin & Young, unpublished) it was found that blood infected with Plusmodium yoelii, a rodent parasite with a predilection for immature erythrocytes, incorporated both purines and pyrimidines at high levels; whereas blood infected with P. chabaudi, which invades mature erythrocytes, incorporated only purines. The
present
work
demonstrates
parasites readily incorporate whether
that
Bubesiu
free purines in vitro,
or not they depend on these base compounds
for synthesising nucleic acids in viva remains to be determined. In many cell lines. if de nova synthesis of purines is blocked, nucleic acid synthesis can proceed via a salvage pathway utilizing exogenous hypoxanthine which is converted to inosinic acid by the enzyme hypoxanthine guanine phosphoribosyl transferase (EC 2.4.2.8) (Szybalska & Szybalski 1962). Similarly the intraerythrocytic stages of plasmodia are apparently unable to synthesize purine nucleotides de nova and depend on the uptake of preformed compounds (Bungener & Nielsen, 1968; Walsh & Sherman, 1968; Walter & Konigk, 1974). If the synthesis of nucleic acids by Babesia is also dependent on such a salvage pathway, as could be inferred by the very ready uptake of hypoxanthine, a possible site for chemical attack is offered. A starting point for such an attack would be the use of drugs such as 6-thioguanine, 8-azaguanine and 6-mercaptopurine, structural analogues of hypoxanthine, whose therapeutic properties have been well explored in the treatment of neoplasia (Connors, 197 1). Studies on the uptake of tritiated nucleic acid precursors by Plasmodium spp. have already been helpful in assessing the effects of various antimalarial compounds; our own studies and those of Chiodini (1973) indicate a similar application for the study of anti-babesial drugs.
CONNORST. A. 1971. Inhibition
precursors
are grateful to Dr. B. F. Sansom for his advice and Drs. N. McHardy and C. Ginger for helpful comments on the manuscript. We also thank Mr. C. Mallinson for carrying out scintillation counts, Mr. I. Jebbett for photography and Mr. D. Hendry for technical
assistance. REFERENCES BERLIN R. D. & OLIVER J. M. 1975. Membrane transport of purine and pyrimidine bases and nucleosides in animal cells. International Review of Cytology 42: 286-336. BUNGENER W. & NIELSON G. 1967. Nukleinsaurenstoffwechsel bei experimenteller malaria. I. Zeitschrifi fiir Tropenmedizin und Parasitoloaie 18: 456-462. BUNG~NER W. & NIELSON G. l&8. Nukleinsaurenstoffwechsel bei experimenteller malaria. II. Zeitschrift fB/ Tropenmedizin und Parasitologic 19: 185-197. CHIODINI P. L. 1973. In vifro culture of Babesiu. Transuctions of the Royal Society of Tropical Medicine and Hygiene 67: 27-28.
of nucleic
of the synthesis of the acids (DNA and RNA) In
Fundamentals of BiochemicalPharmacology (Edited by BACQ Z. M., CAPEK R., PAOLET~~ R. & RENSON J.). Pergamon Press, Oxford.
Cox F. E. G. & YOUNG A. S. 1969. Acquired immunity to Babesia microti and B. rodhaini in mice. Parasitology 59: 257-268. DOLAN T. T. 1974. Mechanisms of pathogenesis experrmental babesiosis. Ph.D. Thesis, Edinburgh.
in
GUTTERIDGE W. E. & TRIGG P. I. 1970. Incorporation of radioactive precursors into DNA and RNA of Plusmedium knonlesi in vitro. Journal of Protozoology 17: 89-96. HOMEWOOD C. A., NEAME K. D. & MOMEN H. 1975. Permeability of erythrocytes from mice infected with Babesia rodhaini. Annals of Tropical Medicine and Parasitology 69: 429-434. IRVIN A. D., BROWN C. G. D., BOARER C. D. H., CRAWFORD J. G. & KANHA~ G. K. 1974. Autoradio-
graphic evidence for the occurrence of cell fusion in cultures of Theileriu-infected bovine lymphoid cells. Research in Veterinary Science 16: 137-142. IRVIN A. D. & STAGG D. A. 1977. Theileria parva: purine and pyrimidine metabolism and the action of folate antagonists in parasitized bovine lymphoid cells. Experimental Parasitology 41: 172-l 85. LIEU T. S., HUDSON R. A., BROWN R. K. & WHITE B. C. 1971. Transport of pyrimidine nucleosides across human erythrocyte membranes. Biochimica et Biophysica Acta 241: 884-893. MAGER J., HERSHKO A., ZEITLIN-BECK R., SHOSHAN~ T. & RAZIN A. 1967. Turnover or purine nucleotides in rabbit erythrocytes. Biochimica et Biophysics Acta 149: 50-58. MORGAN K. & CANNING E. U. 1974. Incorporation of r3Hlthvmidine and VHladenosine bv Eimeria tenella grown-in chick embryos Journal oj Parasitology 60: 364-367. MORZAR~A S. P., BROCKLESBY D. W., HARRADINE D. L. & BARNETT S. F. 1974. Bubesia major in Britain: in-
fectivity Acknowledgements-We
23
of
suspensions
derived
Haemaphysalis punctata nymphs. for Parasitology 4 : 437-438.
from
ground-up
International
Journal
NOWELL F. 1969. The blood picture resulting from Nuttalia (Babesia) rodhaini and Nuttalia (Bubesiu) microti infections in rats and mice. Parasitology 59: 991-1004. OUELLETTE C. A., STROU~ R. G. & MCDOUCALD L. R. 1973. Incorporation of radioactive pyrimidine nucleosides into DNA and RNA of Eimeriu tenella (Coccidia) cultured in vitro. Journal of Protozoology 20: 150-153. PERROTTO J., KEISTER D. B. & GELDERMAN A. H. 1971. Incorporation of precursors into Toxoplasma DNA. Journal of Protozoology 18: 470-473. POLET H. & BARR C. F. 1968. DNA, RNA and protein synthesis in erythrocytic forms of PIasmodium knowlesi. American Journal of Tropical Medicine and Hygiene 17: 672-679. PURNELL R. E., BROCKLFSBY D. W., KITCHENHAM B. A. & YOUP~G E. R. 1976 A. statistical comparison of the behaviour of five British isolates of Babesia divergens in splenectomised calves. Journal of Comparative _.. _.___. Yatho!ogy 116: 609-614.
24
A. D. IRVIN,E. R. YOUNG and R. E. PURNELL
SCHMIDT G., WALTER R. D. & KONIGK E. 1974. Adenosine kinase from normal mouse erythrocytes and from Plasmodium chabaudi: partial purification and characterization. TropenmedizinundParaCtologie25: 301-308. SZYBALSKA E. & SZYBALSKI W. 1962. Genetics of human cell lines. IV. DNA-mediated heritable transformation of a biochemical trait. Proceedings of the National Academy of Science 48: 2026-2034. VAN DYKE K. 1975. Comparison of tritiated hypoxanthine, adenine and adenosine for purine-salvage incorporation into nucleic acids of the malarial parasite, Plasmodium berghei. Tropenmedizin und Parasitologic 26: 232-238.
1.J.P. VOL. 8. 1978
VAN DYKE K., TREMBLAY G. C., LANTZ C. H. & Szus-rKIEWICZ C. 1970. The source of purine and pyrimidines in Plasmodium berghei. American Journal of Tropical Medicine and Hygiene 19: 202-208. WALSH C. J. & SHERMAN I. W. 1968. Purine and pyrimidine synthesis by the avian malaria parasite Plasmodium lophurae. Journal of Protozoology 15: 185-197. WALTER R. D. & KONIGK E. 1974. Hypoxanthine guanine phosphoribosyl transferase and adenine phosphoribosyl transferase from Plasmodium chabaudi, purification and properties. Tropenmedizin und Parasitologic 25: 227-235.