Effects of mitochondrial inhibitors on intraerythrocytic Plasmodium falciparum in in vitro cultures

O'DONOGHUE I T AL.-CHARACTERIZATION OF SARCOCYST'IS SPP.

21. Heydorn, A. 0. & Unterholzner, J. 1983. Zur Entwicldung von Sarcocystis hircicanis n. sp. Berl. Mainch. T i e r i d . Wochenschr..96: 275-282. Gestrich. R.. Mehlhorn, H. & Rommel, M. 22. Heydorn, A. 0.. 1975. Proposal for a new nomenclature of the Sarcosporidia. Z . Parasite&.. 48: 73-82. 23. Kreutzer, R. D. & Sousa. 0. E. 1981. Biochemical characterization of Trypanosom spp. by isozyme electrophoresis. Am. J. 7rop. Med. Hyg., 30: 308-317. 24. Levine, N. D. & Tadros. W. 1980. Named specits and hosts of Sarcocystis (Protozoa: Apicomplexa: Sarcocystidae). Syst. Parasitol.. 2: 4 1-60. 25. Lewontin,R C. 1974. TheCeneiicBasisofEvolutioMlyChnnge. Columbia Univ. Ress, New York. 26. Mehlhorn, H. & Scholtyseck, E. 1973. Elektronenmikroskopische Unlenuchungen an Cystcnstadien von Sarcocystis tenella aus der Oesophagus-Muskulatur des Schafes. Z . Purasitenkd.. 41: 29 1-3 10. 27. Mehlhorn,H., Heydorn, A. 0.& Gatrich, R. 1975. Licht- und elektronenmikroskopishe Untersuchungen an Cysten von Sarcocystis ovicanis Heydorn et al. ( I 975) in der MuskuJatur von Schafen. Z. Parasitenkd., 48: 83-93. 28. 1975. Licht- und elektronenmikroskopische Untmuchungen an Cysten von Sarcocystis fusi/ormis in der Muskulatw von U b c r n nach experimenteller lnfektion mil oocysten und Sporocysten der grossen Form von Isospora bigemina des Hundes. 1. Zur Entstehung der Cyste und der "Cystenwand." Zentralbl. Bakm'ol. Mikrobiol. Hyg. I Abt. Orig. A, 232: 392-409. 29. Mehlhorn, H., Hartley, W.J. & Heydorn, A. 0. 1976. A comparative ultrastructural study of the cyst wall of 13 Sarcocystis species. Protistologica. 12: 45 1-467.

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30. Moult. L.T. 1886. Psorospermies du tissu rnusculairc du mouton. Bull. Mem. SOC.Centr. Med. Vet.. 40: 125-129. 1888. Dcs sarcosporidies et de leur frtquena, princi31. palement chez les animaux de boucherie. Soc. Sci. Arts, Vitry-le-Franqois. 14: 3-42. 32. Peters, W. 1981. Technological advances in protozoal taxonomy and systematics, in Canning, E. U., ad., Parasitological Topics. Allen Press, Lawrence, KS, pp. 200-209. 33. Powell, J. R. 1975. Protein variation in natural populations of animals. Evol. Biol., 8: 79-1 19. 34. Railliet, A. 1886. Psorospermies g&ntes dans I'oesophage et les muscles du mouton. Bull. M h . Soc. Centr. M a . Vet., 40: 130134. 35. 1886a. [Miescheriatenella.] Bull. MPm. Soc. Centr. Ma. Vet., 40: 130. 1886b. Elements de zoologie d d i c d e el agricole. As36. selin et Houseau, Paris. 37. Rommel. M.,Heydom, A. 0.& Gruber, F. 1972. €%itrage zum Lebcnszyklus der Sarlrosporidien. I. Die Sporozystc von Sarcocystis tenella in den Fiizes der Katze. Berl. Mainch. Tierarztl. Wochenschr..

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38. Shirley. M. W. 1978. Electrophoretic variation of enzymes: a further marker for genetic studies of the Eimeria. Z. Parasitenkd., 57: 83-87. 39. Viles, J. M.& Powell, E.C. 1976. The ultrastructure of the cyst wall of a murine Sarcocystis. Z. Parasitenkd.. 49: 127-132. 40. Wenyon, C. M. 1926. Protozoology. Baillih. Tindall & Cox, London. Received 28 V 85: accepted 9 VIII 85

J. frcfoxaol.. 3x1). 1986. pp. 121-125 0 I986 by chc Society of Rotoualogim

Effects of Mitochondria1 Inhibitors on Intraerythrocytic Plasmodium falciparunt in In Vitro Cultures' H. GINSBURG,* A. A. DIVO, T. C. CEARY. M. T. BOUND, d J. B. JENSEN *Lkparfment of Biological Chemistty, Institue of Liye Sciences, The Hebrew University, Jerusalem 91 904. Israel and Department of Microbiology and Public Health, Michigan State University, East Lunsing. Michigan 48824

ABSTRACT. Malarial parasites infecting mammalian hosts are considered to be homolactate fermenton at their asexual intraerythrocytic developmental stage; however, existing ultrastruaural and biochemical evidence suggest that their acristate mitochondna could be involved in energy metabolism. In the present study, inhibiton of mitochondrial function includingcompounds which act on NADH and succinate dehydmgenases, electron transport and mitochondrial ATPase, as well as uncouplcn, were found to inhibit the growth and propagation of the human parasite Plasmodiumfalcipmm in in vitro cultum at concentrations that specifically affect mitochondrial functions. Direct measurement of parasite protein and nucleic acid synthesis in synchronized cultures showed that throughout the patasitc life cycle both processeswere inhibited, the latter proccss being more sensitive. These mults strongly suggest that intraerythrocytic malarial parasites require mitochondrial energy production.

M

ITOCHONDRIAL function in the asexual erythrocytic

stages of mammalian malaria parasites is poorly understood. Plasmodium spp. lack a full complement of citric acid cycle enzymes (28) and it i s generally accepted that these organisms derive most, if not all, o f their energy requirements via glycolysis (25,27). Acristate mitochondria with cytochrome oxidase activity, however, h av e been found in all species investigated (26). and it has been suggested that the exclusive role of the organelle in these cells is to serve as an electron disposal mechanism in de no v o pyrimidine biosynthesis ( 1 2, 15).

I

This work was supported in part by grants from the UNDPNorld

BanWHO Special Programme for Research and Training in Tropical DiscaSeS.

Several lines o f evidence suggest, however, that parasite mitochondria may have additional functions, possibly including energy metabolism. Inhibitors o f mitochondrial protein synthesis a re potent antimalarial agents (2,9). and we have recently demonstrated that the mitochondrion of P.fakiparum actively maintains an electron transport-dependent transmembrane electrical potential and undergoes a complex pattern of growth a nd development throughout the asexual erythrocytic cycle (5). There are also indications that rodent malaria parasites possess a n electron transport chain that operates in energy-consuming processes (1 8, 29) a n d that most parasite species tested require a nd utilize 0, and assimilate CO, (28). In the present study we show that different inhibitors that are relatively specific for various aspects o f mitochondrial function efficiently impair parasite viability a n d anabolism. Thus, in

122

J. PROTOZOOL..VOL. 33. NO. I , FEBRUARY 1986

TABLE I. Inhibition of parasite growth by inhibitors of mitochondrialfunction Parasite strain.

Rotenone

1.7. lo-'

TrF Oligomycin

8.5.10-'

Rhodamine 123 Janus Green DCCD Quercetin Antimycin A, HQNO

CNSHAM CCCP A23 I87

1.6.10-'

2.0.10-6 3.3. lo-' 2.8.10A6 4.7.10a6 I .4. lo-' 6.3.10-7 2.0.10-7

3.9.10-7 1.6.10-10 2.3.10-6 4.4,10-6 7.5. lo-' 1.9.10-' 1.8. loe6

2.7.10-7 4.5. lo-"

4.4- 10 - 1 9.7.10-11 2.0.10-6 5.5.10-6 5.7.

6.3.10.-7

4.5. lo-' 1.6. lo-' 4.3.10-7

3.3.10-7

a Parasites of different strains were cultivated in candle j a n for 48 h in culturt media containing increasing concentrations of the different inhibitors and ['mhypoxanthine. The uptake of radiolabel into parasite mattrial was determined and compared to label-incorporation in control cultures to assess the extent of inhibition. result^ arc presented in terms of IC, values (molar concentration needed to cause 50% inhibition) derived according to Dcsjardins et al. (4).

agreement with previous reports, it is suggested that the mitochondrion is critical for parasite survival, and its possible interplay with energy metabolism cannot be discounted. MATERIALS A N D METHODS Cultures of Plasmodium falciparum were routinely maintained in candle jars (20); strains used were FCR,,,, FCC,, FCR,, FCR,, Vietnam Smith ( V N S ) (lo), and FCMSU,/Sudan (6). Effects of drugs on parasite viability in 48-h assays were determined with [Wlhypoxanthine incorporation as described (5. 11). Values of ICx, represent drug concentrations causing 50% decrease in label incorporation compared to control and were determined according to Desjardins et al. (4). Experiments were initiated with parasites synchronized to the schizont stage (23) at I-2% parasitemia. Short term experiments used sorbitol-synchronized parasites (23) and were run in 96-well flat-bottom plastic microtiter plates (Flow Laboratories Inc.). Wells received 100 pl of cultures at 1 6 2 0 % parasitemia, 3-5% hematocrit. in RPMI-I640 (Grand Island Biological Co.)supplemented with 10% pooled human A' serum, 25 m M N-2-hydroxyethylpiperazine-K-2cthane sulfonic acid (HEPES) and 2.5 m M NaHCO,. Media contained various concentrations of drugs and 20-50 pCi/ml ['Hlphenylalanine (82 Ci/mmole) or ['HJhypoxanthine ( 1 0 Ci/mmole, both from New England Nuclear Corp.). Incubations were carried out in candle jars at 37°C. At various time intervals, cultures were harvested (Bellco MASH II) onto glass fiber filters and processed for scintillation spectroscopy as described (1 I). Prior to incubation, the total radioactivity in the various systems was estimated on 10-pl aliquots taken from the whole suspension. Radioactivity associated with filters was normalized to the total cpm to correct for experimental errors in diluting and pipetting. Means of triplicates of normalized cpm in the presencc of drug were compared to those of drug-free controls harvested simultaneously, and data are thus expressed as percent inhibition. Inhibitors used (and their targets) were rotenone (NADH dehydrogenase); thenoyltrifluoroacetone (TTF) (succinate dehydrogenase); n-heptyl-4-hydroxyquinoline N-oxide (HQNO), antimycin A, (AA), salicylhydroxamicacid (SHAM), NaN,, and KCN, (electron transport); oligomycin, quercetin, rhodamine 123, Janus Green, and N . ~ - d i c y c l o h e x y l c i m i d e(DCCD)

(ATP synthetase); 2,4-dinitrophenol (DNP), carbonylcyanide nchlorophenyl hydrazone (CCCP), and A23 187 (uncouplers). All reagents and drugs, except rhodamine 123, which was obtained from Eastman Kodak, Rochester, NY,were purchased from Sigma Chemical Co. The A23 187 was a gift From Dr. J. L. Bennett.

RESULTS Data on the effect of mitochondrial inhibitors on the growth and propagation of P. falciparum in culture are presented in Table I. All compounds tested had IC,, values within the range of specific antimitochondrirl effects. Combinations of KCNSHAM, antimycin A,-KCN, rotenone-TTF and rotenone-antimycin A, were also tested and in every case simple additive interactions were found (data not shown). Results in Table I also demonstrate little interstrain variation in sensitivity to the various drugs, none of which was correlated with sensitivity to chloroquine ( 1 9); however, profound variation in susceptibility to KCN was observed. Short term effects of selected inhibitors on parasite protein and nucleic acid synthesis are depicted in Fig. 1. Drug concentrations were somewhat higher than those used in viability assays in order to amplify the effect, but never exceeded those typically used in similar studies. Results show that nucleic acid synthesis is more sensitive to mitochondria1 inhibitors than protein synthesis. Inhibition of hypoxanthine incorporation in ring stage parasites was detected already 30 min after exposure to the drugs and inhibition of protein synthesis after 60 min. Mature parasites were affected earlier, perhaps due to age-dependent increases in permeability of host cell membrane (14) and were also more active metabolically, with ten times higher incorporation of hypoxanthine and five times higher of phenylalanine, compared to rings. The time course of inhibition by DNP shows an initial temporary inhibition and no effect later on. When serum was omitted from the culture medium, inhibition by this uncoupler was more pronounced and constant throughout the experimental period (data not shown). This phenomenon can be interpreted in terms of the binding of DNP to serum albumin (30). Data shown in Fig. 2 depict the effects of other inhibitors in similarexperimentsonadifferentparasitestrain(FCR,,). Again, only minor differences in the relative inhibition at the different

GlNSBURG ET AL.-MITOCHONDRLAL INHIBITORS ON P. FALCIPAR UM

123

growth stages were observed, and protein synthesis was less

sensitive than nucleic acid synthesis. The most effective inhibitor was TTF, implicating succinate and succinate dehydrogenase as critical components of mitochondria1 function (1). Timtion of the effect of l T F in 2-h exposures on the FCMSUJ Sudan strain revealed IC,,, values of 4.8 x M and