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L; \I ' 11 x Eggs; . 1:”: “ .. “‘ ‘7 ‘7 7'. I a“??? $17737 “13 “$7: “Mist 1797‘! 39.77%- KEWW‘ZM ’ . .. M’vwa-Efiguu :1‘ 2:4;wa 1 Vary” '17';"""‘.1MMEVE-“.L‘fi.gj."~~ 3'}: 4.1. @773 1:: W: _ 7 ("'1' ' : , :." {“31“ ' “Rmfifiyflgl‘dy‘l? “$7.3 :Y;‘::1L: ‘ II Slug ‘1?»ka 7', , , --_‘ .~- ,~. .3: ”f! EVE? {g’KhJ‘ ‘1‘ . w LVCHVSVEV‘ ;~ . Riga v '2 ;;;; I) ' This is to certify that the dissertation entitled THE MITOCHONDRION OF PLASMODIUM FALCIPARUM AS A SITE FOR CHEMOTHERAPEUTIC INTERVENTION presented by ALAN ANDREW DIVO has been accepted towards fulfillment of the requirements for Ph AD - degree in My DateXfiZefa/é 0,73, / 755’ MS U is an Affirmative Action/Equal Opportunity Institution 0-12771 MSU LIBRARIES m RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. THE MITOCHONDRION OF PLASMODIUM FALCIPARUM AS A SITE FOR CHEMOTHERAPEUTIC INTERVENTION BY Alan Andrew Divo A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Microbiology and Public Health 1985 Bea/toe»? ABSTRACT THE MITOCHONDRION OF PLASMODIUM FALCIPARUM AS A SITE FOR CHEMOTHERAPEUTIC INTERVENTION BY Alan Andrew Divo Continuous cultivation of Plasmodium falciparum presently requires the complex medium RPMI 1640 supplemented with human, bovine, or rabbit serum. Metabolic studies, identification and characterization of potential antimalarial drugs, and radiolabeling macromolecules are difficult using such a complex medium. The low—molecular- weight, water-soluble nutrients required for parasite growth in yitrg have been determined. Using exhaustively dialyzed human serum and reformulating the culture medium it was found that only 7 of 19 amino acids and l of 11 vitamins are required for continuous parasite growth. Although of questionable physiological significance, recent experiments demonstrating the inhibitory effects of 7OS-specific ribosome inhibitors and an exquisite sensitivity to riboflavin antagonists indicate that mitochondria are critical for survival of the parasite. The lack of TCA cycle enzymes have left the classical function of the mitochondrion in g. falciparum in doubt. The Alan Andrew Divo antimalarial activity of clindamycin was reported to be 02— and time-dependent. This observation has been extended to a variety of antibiotics whose only common mechanism of action is the inhibition of protein synthesis on 708 ribosomes. In 96 h incubations, potency was increased by a factor of up to 106 over the first 48 h period and by a factor of up to 104 in 15% 0 versus 1% O . The effects were specific; a wide 2 2 variety of antimalarial drugs lacked both 02— and time- dependent toxicity. The effects of the 8OS-specific protein synthesis inhibitors cycloheximide and anisomycin were not affected by oxygen. The vital dye rhodamine 123 (Rh123) was used to probe the functional status of the mitochondrion within the asexual, intraerythrocytic stages of g. falciparum. Using epifluorescence microscopy the development of a single mitochondrion has been followed through the intraerythrocytic cycle. The effects of uncouplers, ionophores, and inhibitors of ATPase and electron transport activities were examined by monitoring Rh123 accumulation and retention. In most cases our results were similar to those obtained in other eukaryotic cells. The importance of the mitochondrion in g. falciparum has been exemplified further by the toxicity exhibited by those agents which affected Rh123 retention and by that of Rh123 itself. To Christine and Erich ii ACKNOWLEDGMENTS I wish to express my sincere gratitude to Dr. James Jensen for providing a pleasant and stimulating environment in which to work. I would also like to thank my committee members for their participation: Drs. Williams, Bennett, Miller, and Corner. I am also greatful for the friendships I have had while here at MSU. I will especially miss John, Mike, and Tim. Above all others I thank my wife, Christine, and my son, Erich, for their encouragement and support. For their sacrifices we shall benefit as a family in the future. iii TABLE OF CONTENTS PAGE LIST OF TABLES.... ......... ............................ Vi LIST OF FIGURES ............. . ......... ................. vii INTRODUCTION........................ ..... ........ ..... . 1 LITERATURE REVIEW..................... ............ ..... 6 Biochemistry of the amino acids in erythrocytic Plasmodium ............... ............... 6 Protein synthesis ............................ 6 Biosynthesis of amino acids.................. 7 Digestion of host cell hemoglobin............ 7 Exogenous amino acids........................ 8 Amino acid metabolism..... .......... ......... 9 Biochemistry of the water-soluble vitamins in erythrocytic Plasmodium ................. 10 Folates... .......... . ........................ 10 Pantothenates ...................... .......... 11 Riboflavin. ...... .............. ............ .. 12 Pyridoxine, thiamin, nicotinamide, biotin, and ascorbic acid....................... 13 Biochemistry of the carbohydrates and its relationship to mitochondrial function in erythrocytic Plasmodium. ............. 14 Introduction... .............. ................ 14 Transport and metabolism..... .............. .. 14 Oxygen utilization and electron transport.... 17 Bibliography.... ................. ................. 20 CHAPTER 1. NUTRITIONAL REQUIREMENTS OF PLASMODIUM FALCIPARUM IN CULTURE. I. EXOGENOUSLY SUPPLIED DIALYZABLE COMPONENTS NECESSARY FOR CONTINUOUS GROWTH.. ..... ... .......... . ................. ...... ..... 36 Abstract .......... .............. ....... ........... 36 Introduction.................... ...... .. ..... ..... 38 Materials and methods ....... .............. ........ 39 Parasites ..... . ..................... .... ..... 39 Serum and erythrocytes ......... .............. 39 Media preparation ......................... ... 40 Assessment of parasite growth .......... ...... 41 Amino acid incorporation... ............ ...... 42 Results. ...................................... .... 43 Discussion................... ....... .............. 52 iv Acknowledgment................. .......... ......... Literature cited.................................. CHAPTER 2. OXYGEN- AND TIME-DEPENDENT EFFECTS OF ANTIBIOTICS AND SELECTED MITOCHONDRIAL INHIBITORS ON PLASMODIUM FALCIPARUM IN CULTURE......... ..... ......... Abstract............... .................... ....... Introduction.......... ................... .. ....... Materials and methods ........... ... ............... Results ............... . ............. .............. Discussion.......... ..... . ........... . ...... . ..... Acknowledgment...... ..... ......................... Literature cited..... ...... . ............ . ..... .... CHAPTER 3. THE MITOCHONDRION OF PLASMODIUM FALCIPARUM VISUALIZED BY RHODAMINE 123 FLUORESCENCE............... Abstract ..... .............................. ...... . Introduction...................... ..... ........... Materials and methods .......... ....... ..... ....... Parasites. ........ .... ........... .. .......... Rh123 staining............. ..... ............. Treatment with mitochondrial inhibitors...... Epifluorescence microscopy................... Reagents ......................... ............ Results......... ..... . ........ .................... Discussion........................................ Acknowledgments ............. . ................ ..... Literature cited ........... . ............. ......... 59 6O CHAPTER 1 CHAPTER 2 CHAPTER 3 TABLE I II III IV II LIST OF TABLES PAGE Effects of depletion of single amino acids or vitamins from Recon—10D medium on [ thypoxanthine incorporation by the FCR3 isolate of Plasmodium falciparum for $5 or 96 h in culture............44 Growth of FCR isolate of Plasmodium I 0 TC 0 a u falc1parum 1% basal medium containing pantothenate and various amino aCidSOOOOOOO0...0.0.0000000000000000045 Growth comparisons of the FCR , FCMSU /Sudan, VNS, and Honduras 638 isolates of Plasmodium falcipardm in RP -10 and minimal medium...............49 Incorporation of [3H]amino acids into proteins of the FCR3T isolate of Plasmodium falciparum Suring 48-h exposureSOOOOOOOOOOOOOOO......OOOOOOOSO Growth of the FCR C isolate Plasmodium falciparum in bass? medium supplemented with pantothenate, and variuos amino aCidS’ and PBS or PBSOOOOOOOOOOOOOOOOSl Influence of time and oxygen on potency of antimalarial agents in culture of P. falciparum (FCR3TC isolate)..........74 Effects of mitochondrial inhibitors on the accumulation or retention of Rh123000000000000.0.0.00000000000000105 vi CHAPTER 1 CHAPTER 2 CHAPTER 3 FIGURE 1 LIST OF FIGURES PAGE Growth curves for the FCR isolate of Plasmodium falcipgggm in culture..........................48 Concentration response curves describing the inhibition of P, falciparum by clindamycin and related derivatives.................73 Concentration-response curves describing the effects of other 708 ribosome inhibitors on P. falciparum in culture...............76 Concentration-response curves describing the effects of the 805 ribosome inhibitors anisomycin and cycloheximide on P. falciparum......79 Concentration—response curves describing the effects of the mitochondrial inhibitors Janus Green, rhodamine 123, and antimycin A on P. falciparum in culture...............81 Mitochondria of P. falciparum stained with rhodamine 123.................102 vii INTRODUCTION Malaria due to the obligate intracellular parasite Plasmodium falciparum continues to be a disease causing both high morbidity and mortality (1). The once promising pros- pect of world eradication of malaria has all but vanished. The causes for the continuing decline in the world's malaria situation include both social and biological factors (2). The reduced susceptibility of the anopheline vector to insecticides (3) and the increasing spread of chloroquine resistant strains of P. falciparum (1, 4) have stimulated a considerable effort toward the identification of new antimalarial drugs. Except for the antifolates, most of this effort has been directed toward drugs which are modifications cfif the quinoline-containing antimalarial agents quinine and chloroquine (5, 6, 7) which may not escape the problem of resistance that plagues the proto- types. The seriousness of the situation would be difficult to overestimate (4, 8, 9, 10). Although a tremendous effort has been made to under- stand the immunology of malarial infection in the hopes of developing a vaccine, many obstacles exist (11-15). Since the prospects for a safe, effective, and commercially feasible 'vaccine against .B° falciparunu remain. uncertain, chemotherapy 5&3 the primary defense against this disease. The problem of drug resistance demands novel approaches to chemotherapy, since resistance may develop rapidly against drugs which are simply modifications of currently available compounds. With the parasite readily available for study (16-20), the identification and characterization of new antimalarials has been facilitated considerably. As well, a much more comprehensive understanding of the basic biochemical and physiological processes required for the survival of this organism may now be described. If approached rationally, the inefficient practice of randomly screening compounds for antimalarial activity will give way to the design of new site-specific drugs. Continuous cultivation of P. falciparum was originally achieved. using' a :nutritionally complex: medium, RPMI 1640 (RP), supplemented with human serum (16, 17). Although some success has been achieved in characterizing dialyzable (21, 22) and nondialyzable (22, 23, 24) serum factors essential for the in yitrg maintenance of this organism, little prog- ress has been reported in the analysis of the components of RP which are necessary for continuous culture. Employing such a complex medium complicates metabolic studies, the identification and characterization of potential antima- larial drugs, and radiolabeling of macromolecules in the parasite. Numerous attempts made tx> discover the nutritional requirements of Plasmodium spp. (24-54) illustrate the interest generated by this topic. However, most of these studies were done _i_n vivo; the effects of nutrient depletion on the host were difficult to separate from those on the parasite. Studies previously conducted in yitrg used parasites that could not survive in the culture system and must be considered somewhat suspect. The development of techniques for continuous culture of P. falciparum has allowed us to determine the requirements for some of the exogenously supplied water-soluble vitamins and amino acids. Although exhaustive dialysis eliminates low- molecular—weight water-soluble nutrients from human serum, it is difficult to eliminate such compounds from intact erythrocytes, which may provide the parasite with an adequate supply of some nutrients (55, 56). Therefore, only the amino acids and vitamins that are exogenously supplied by serum or medium (RP) were examined. The contribution of erythrocyte nutrients present endogenously was not examined in the present study; but was examined iJi a later study employing a series of vitamin antagonists (57). Interestingly, the studies utilizing vitamin analogs revealed that P. falciparum is extremely sensitive to analogs of riboflavin and sensitive to the nicotinamide analog, 6—aminonicotinamide (57). These vitamins are important for mitochondrial functions and malarial parasites have been shown to possess flavin-linked dihydroorotate dehydrogenase (74, 75) and NADH dehydrogenase and NADH oxidase activities (187, 188). Additionally, antimalarial activity has been demonstrated for some of the antibiotics, many of which are active against P. falciparum in vitro at relevant pharmacological concentrations (58, 59). The active antibiotics have demonstratable antimalarial effects in animal models (60-63), and tetracyclines (64-68) and clindamycin (69—72) have been shown to be effective against P. falciparum in humans. Although no data are available on the mechanism(s) of antimalarial action of these drugs, it has been suggested that they act on parasite mitochondria, which like other eukaryotic mitochondria, probably contain 708 ribosomes (58, 73). However, the acristate morphology (76, 77) and the apparent lack of most TCA cycle enzymes (42, 78) have left the classical function of the mitochon— drion in P. falciparum in doubt. Since definitive studies have yet to determine the metabolic role (if any) of this organelle in the erythrocytic stages of this parasite (42), it is difficult to directly test the hypothesis that an antimalarial drug acts at the mitochondrial level. It has been shown that the antimalarial potency of some antibiotics is influenced by the duration of exposure in culture (58, 79) and, for clindamycin, by oxygen tension (79). The mitochondrial toxin hypothesis was indirectly tested by using these two variables to compare the effects of various antibiotics, mitochondrial inhibitors, and other drugs on development of the sexual, intraerythrocytic stages of P. falciparum in 31539. The functional status of the mitochondrion was also probed using the vital dye rhodamine 123 (Rh123). This cationic fluorescent dye accumulates specifically in negatively charged subcellular compartments, such as active mitochondria (80-82). Using epifluores- scence microscopy the development of the mitochondrion of P. falciparum has been described over the course of the asexu— al, intraerythrocytic cycle. The effects of uncouplers (83— 85), ionophores (86), and inhibitors of ATPase activity (87, 88) and electron transport (89, 90) were examined by monitoring the accumulation and retention of Rh123. The literature review has been divided into three sections: 1) biochemistry of the amino acids in erythrocytic Plasmodium, 2) biochemistry of the water-soluble vitamins in erythrocytic Plasmodium, 3) biochemistry of the carbohy- drates and its relationship to mitochondrial function in erythrocytic Plasmodium. The review concentrates on the asexual, intraerythrocytic cycle; the sexual cycle or gamatogony will not be reviewed. As well, the discussion has been directed more towards mammalian than other species of malarial parasites. LI TERATURE REVI EW Biochemistry of the amino acids in erythrocytic Plasmodium Protein synthesis. The erythrocytic stages of the malaria parasite acquire amino acids from three sources. They arise from either £2.2222 synthesis, digestion of host cell hemoglobin, or the uptake of exogenous amino acids present in the plasma of the host. Protein synthesis in plasmodia appears to be typically eukaryotic. The cytoplas- mic ribosomes and their associated RNA from both human and nonhuman malarial parasites are typically protozoan by a variety of methods such as base ratio analysis (91, 94), velocity sedimentation (91-93, 95), and gel electrophoresis (93, 94, 96, 97). Like other eukaryotic organisms, in the absence of magnesium, the 808 ribosomal particle dissociates into the large 59-608 and small 40-445 subunits (91, 93, 95). The high molecular weight 24.2-255 and 16.6-17.48 (92, 94, 96, 97) RNA species are typically protozoan by base composition (G + C content 37-43 mole %; 92, 94, 96, 97), sedimentation velocity, and molecular weight (92-94, 96-97). Inhibitors of eukaryotic 80$ ribosomes, such as cyclohexi— mide (58) and anisomycin (59), have antimalarial effects against P. falciparum in vitro. Mitochondrial ribosomes of the malarial parasite have yet to be examined directly. The synthesis of proteins in highly synchronizied cultures of P. falciparum varies both quantitatively and qualitatively as the erythrocytic parasite matures from the ring to schizont stage (98-101). Biosynthesis of amino acids. Malarial parasites synthe— size glutamic and aspartic acids, methionine, and alanine by C02 fixation (102-105). Only small amounts of amino acids synthesized g3 novo by plasmodia are in parasite proteins. Digestion of host cell hemoglobin. Host cell hemoglobin has been shown to provide the erythrocytic malarial parasite with most amino acids required for protein synthesis (42, 105, 106). When radiolabeled RBC's were transfused into E. lophurae or P. knowlesi infected hosts, parasites which subsequently developed in the labeled cells were found to have incorporated labeled host cell amino acids (106, 107, 108). Hemoglobin determinations have indicated that a large proportion of host cell hemoglobin is destroyed over the period of parasite development (105). A number of genetic conditions which affect hemoglobin (109-112) prevented or limited the ability of P. falciparum to survive intracellu- ularly. As well, compounds that covalently modify hemoglo- bin, but do not interfere with normal 0 transport, 2 inhibited the growth of P. falciparum in vitro (113). These compounds were most inhibitory against mature parasites, indicating that the requirements for hemoglobin-derived amino acids are greatest during the later stages of intraerythrocytic development. Electron micrographs have illustrated that hemoglobin is first phagocytized by a specialized organelle, the cytosome, forming a phagolysosome (114, 115). Fusion of the phagolysosome with a primary lysosome occurs to form the parasite food vacuole. Enzyme analysis has indicated that hemoglobin probably first autooxidizes and is then acted on by proteases (105, 116). All species of malarial parasites appear to have one or several proteases (116-124), some of which have been purified and are able to degrade hemoglobin (118, 121-124). The released products of hemoglobin diges— tion, hematin and associated protein fragments, accumulate and form aggregates of the malarial pigment, hemozoin. The exact composition of hemozoin has remained uncertain; isolation procedures appear to affect the degree to which proteins are associated with hematin (105) Exogenous amino acids. The importance of exogenously supplied methionine was recognized during early attempts to cultivate P. knowlesi (26); later both methionine and isoleucine were found to be essential for the growth of both mammalian and avian malarial parasites (49-54). Glutamine has been found to be beneficial for in 21359 development of £3 knowlesi (125). Incorporation studies ihave indicated that most exogenously supplied amino acids are utilized to some degree by the malarial parasite (54). These investiga- tions have demonstrated that isoleucine and nethionine are consistently taken up by mammalian species. Except for these two amino acids, there appears to be little correla- tion between the rate of amino acid uptake by the parasite and the amino content of the host cell hemoglobin. For the avian parasihe, P. lophurae, amino acid incorporation was similiar to that of the mammalian species, with the exception of a higher rate for proline incorporation (42). Data from transport studies have shown that malarial parasites cause an alteration of host cell permeability to amino acids (126, 127). Amino acid metabolism. It has been postulated that glutamate may be oxidized by a parasite-specific NADP— glutamate dehydrogenase coupled to the enzymes of the citric acid cycle to provide an ancillary energy source in the avian. malarias (105). Erythrocyte-free .P. lophurae was found to oxidize glutamate to C02 (106). Glutamate dehydro- genase has also been identified in rodent malarias (128, 129) and is thought to be involved in NADP+ reduction (105). The conversion of glutamate to alpha-ketoglutarate may be a primary mechanism for regeneration of the NADPH needed for reductive synthesis. All Plasmodium species synthesize pyrimidines g§_runn> (42, 75) and therefore, are likely to require substantial amounts of glutamine and aspartic acid. 10 Biochemistry of the water-soluble vitamins in erythrocytic Plasmodium Folates. Numerous experiments and observations (24-33) have demonstrated that the plasmodial parasites synthesize folate cofactors g3 pogo, requiring only p-aminobenzoic acid (PABA) for folate synthesis. The importance of PABA was first recognized using sulfonamides to treat human malaria infections (31). PABA was found to reverse the effects of sulfanilamide on P. gallinaceum in vitro (31). The growth of P. knowlesi in vitro required the addition of PABA to the culture medium (26); inhibition of growth by sulfadiazine was reversed by PABA. Parasitemias were suppressed in P. berqhei— and P. cynomolgi-infected animals maintained on milk. diets (28, 29); dietary' PABA reversed. this effect. Pyrimethamine and sulfadoxine act synergistically against P. falciparum (130). Testing P. falciparum in. vitro has revealed that significant differences in potency exist between sulfonamides ‘whichl are essentially' equipotent. as antibacterials (131). The dihydropteroate synthetase of P. falciparum may have significant differences in sensitivity to inhibitors compared to the bacterial enzyme. Differen— tial screening of the PABA analogs against bacteria and P. falciparum in vitro may identify compounds which are selectively toxic against only the malarial parasite. Similar results were obtained using P. falciparum infected Aotus monkeys (c.f. 31). Enyzmes associated with 11 g; novo folate biosynthesis have been identified in a number of Plasmodium species (c.f. 31). Exogenously supplied fo- lates were thought not to be utilized by the malarial para- sites, but recently, enhanced activity against P. falciparum was reported for PABA antagonists and dihydrofolate reduc— tase (DHFR) inhibitors when tested in media which lack PABA and folic acid (130, 132, 133). The potency of aminopterin and pyrimethamine against P. falciparum was decreased by increasing PABA or folic acid concentrations in the culture medium (130, 133). Other Plasmodium lack folate reductase (31); if true for P. falciparum, it is unlikely that folic acid can be used directly by this parasite and drug reversal is probably due to contaminating PABA or folinic acid. Folinic acid is already reduced and other eukaryotes can use folinic acid to partially bypass the blockade caused by DHFR inhibitors (134). Pantothenates. The importance of exogenously supplied pantothenate was first noted during early attempts to culture P. lophurae in duck erythrocytes (34). When calcium pantothenate was added.tx>.a complex medium infectivity was demonstrated after extended periods of in XEEEE incubation, but without added pantothenate infectivity was quickly lost. A dietary deficiency of pantothenate markedly inhibited development of P. gallinaceum in chickens (35). In short— term experiments with “P. falciparum and .P. coatneyi ‘in vitro, it was shown that antimetabolites of pantothenate inhibited parasite development (36,39). In studies using 12 erythrocyte-free P. lophurae, the one species of malarial parasite that has been cultured free from its host erythro- cyte (36), it was found that coenzyme A (CoA), but not pan- tothenate, had a beneficial effect on extracellular parasite development (37, 38). Furthermore, precursors of CoA, such as pantothenate, were ineffective (135). Enzymes associated with CoA synthesis could not be identified in P. lophurae, although they were readily detectable in the duck RBC (40, 41). It was therefore concluded that the malarial parasites require preformed CoA from the host erythrocyte. Riboflavin. Although experiments in vivo (44, 46, 47) indicated that riboflavin is important for the survival and multiplication of both human and animal malarias, its omission from the culture medium did not detract from the growth of P. knowlesi in yiggg (30). Very little has been determined about the functional roles of FAD and FMN in the metabolism of Plasmodium. It was suggested that riboflavin acts indirectly by raising glutathione levels within the host red cell (54); glutathione reductase is a flavin-linked enzyme. Infected cells have been found to be sensitive to oxidant stress (136) and thus must maintain adequate reduced glutathione concentrations to support parasite growth. Malarial parasites also synthesize pyrimidines d3 pogo (42) which requires the flavin-linked enzyme dihydroorotate dehydrogenase (74). Although not conclusively demonstrated, we predict that plasmodia require riboflavin for growth. 13 Pyridoxine, thiamin, nicotinamide, biotin, and ascorbic acid. A limited number of reports exist on the requirements of these vitamins for Plasmodium growth. Detailed experi- ments defining pmecise biochemical roles for these growth factors have not been reported. Most experiments have been conducted in yiyg, where the cause-and-effect relationships are most difficult to analyze. Dietary deficiencies of thiamin (B and pyridoxine (B6) in rats (44) and l) nicotinamide in chicks (137) inhibited multiplication of P. berghei and P. lophurae, respectively. Pyridoxal kinase has been suggested to be provided by the host erythrocyte (43, 45). Nicotinamide was found to favor the survival of erythrocyte "free" P. lophurae in vitro (44). When examined in vivo anu ascorbic acid deficiency suppressed the development of 1P. knowlesi (42), but ‘when omitted from culture medium no apparent effect was observed (30). Although the cultivation of P. falciparum (16) was accomplished almost 10 years ago, to date, neither the exogenous or endogenous vitamin requirements have been investigated in vitro. 14 Biochemistry of the carbohydrates and its relationship to mitochondrial function in erythrocytic Plasmodium Introduction. Knowledge cflf the fundamentally inter- related processes of glucose utilization and energy trans- duction remains speculative for P. falciparum. The earliest studies of plasmodial biochemistry concentrated on carbohydrates; but many of these studies have been justifiably criticized for numerous reasons (42, 55, 138). Techniques were inadequate for maintaining parasite viability ‘in. ‘yiggg; thus metabolic parameters were determined on dying parasites. Since these cultures contained host contaminants, such as immature erythrocytes, platelets, and leukocytes, which all contributed to the observed results, it was impossible to dissect the metabolism of the parasite from that of the other cells. Methods employed for removing unwanted cells from parasit- tized blood varied in their degree of success (139, 140, 141); even "free" parasites prepared by saponin lysis remained contaminated with host cell membranes (138, 142). For these reasons many (Hi the earlier reports on carbohydrate metabolism were in error. Transport and metabolism. Intraerythrocytic stages of Plasmodium require simple sugars as an energy source; they do not store glycogen as a carbohydrate reserve (139, 143). This requirement was appreciated in early attempts to culture malarial parasites in vitro. In 1912, it was 15 demonstrated that glucose was required for in vitro devel- opment of P. falciparum and P. vivax (144). Infections of P. gallinaceum in chickens were more severe when the animals received intravenous injections of glucose (145). Studies in vitro have indicated that substrate utilization is species specifrc, but that all Pdasmodium utilize glucose (42). Infected erythrocytes consume dramatically more glucose than uninfected cells (138, 139, 143, 146-152). To accomodate this increased demand the infected erythrocyte membrane undergoes permeability changes (147, 149-151); simple diffusion and carrier-mediated transport are affected. Although erythrocytic glucose metabolism varies between species, similar pathways exist within the mammalian and avian groups of malarial parasites. In general, mammalian species incompletely oxidize glucose producing organic acids, predominantly lactate, as well as neutral volitiles, and small amounts of succinate, and keto acids (139, 141, 146, 148, 152-159). Avian plasmodia oxidize glucose more completely to yield CO and organic acids (160-163). All 2 Plasmodium species possess the glycolytic enzymes typical of the Embden-Meyerhof pathway (143, 154, 164-172), and that only the avian species appear to possess the required enzymes for the TCA cycle (163, 173-178). Electron micro- scopy has shown that the avian parasites have cristate mito- chondria (179), whereas in mammalian species the mitochondria are typically acristate (180). It has been 16 suggested that in addition to the conventional glycolytic pathway, malarial parasites may' be capable of enzymatic cleavage of pyruvate to yield acetate and ATP (42). The simian parasite, P. knowlesi, was not only shown to oxidize glucose, but also lactate and pyruvate (153). Using labeled lactate, radioactivity was found in phosphoenolpyruvate (PEP), 3-phosphoglycerate, and malate. A possible reaction sequence giving rise to these products would involve the conversion of lactate into pyruvate and pyruvate into oxalacetate, the latter being catalyzed by py— ruvate carboxylase. Oxaloacetate can be converted into PEP in the presence of GTP by the action PEP carboxykinase. The two enyzmes participating in the CO2 fixation reaction have been partially purified from P. berghei (181, 182). Direct transformation of pyruvate into PEP by pyruvate phosphate diphosphotransferase in the presence of ATP and inorganic phosphate occurs in the protozoan Entamoeba histolytica which lacks pyruvate kinase (183). Malate could be derived from oxalacetate by the action of malate dehydrogenase; this enzyme has been detected in P. berghei (173, 184). Conversion of lactate into PEP may be significant since this compound may serve as a source of ATP for the parasite. The growth of cell-free P. lophurae was shown to be favor- ed by the addition of a mixture of red cell extract contain- ing either ATP and pyruvate of ADP and PEP (172). It may be possible that some of these compounds which normally do not cross the cell membrane enter the parasite by pinocytosis or 17 related mechanisms. It has been proposed that the parasite membrane may be extremely leaky and permit the passage of large molecules (42). It appears that lactate metabolism in malarial parasites is more complex than previously assessed. Oxygen utilization and electron transport. Early studies demonstrated that all plasmodia take up 0 but the 2’ extent of O2 tujiization by the parasite was difficult to interpret due to the presence of host cell contaminants (42, 55, 138). Attempts to culture P. lophurae (80) and P. knowlesi (26, 33) indicate that growth was favored at reduced 0 2 tensions and that high concentrations of 0 were 2 detrimental to parasite survival. The Trager and Jensen method (16, 17) has been used to determine that P. falciparum is an cmdigate microaerophile, optimum parasite 2, 1% O2 and the balance N2 (186). Growth was inhibited under anaerobic conditions or when 02 was greater than 21%. Oxygen utilization, was stimulated in P. knowlesi-infected growth occurred in an atmosphere of 3% CO erythrocytes by glucose, lactate, and pyruvate (153). The infected cells were able to utilize both glucose and lactate as a source of energy, since ATP levels were high in the presence of either of these substrates. In comparison with lactate, pyruvate gave lower respiratory rates and it was also less efficient for maintaining intracellular ATP levels. The reason for this difference has not been examined directly, but it is thought that lactate is converted to pyruvate by lactate dehydrogen- ase, a reaction 18 that would give rise to the reduced nucleo- tide NADH (153). Furthermore, NADH oxidation may be linked to electron transport and oxidative phosphorylation. Al- though experimental evidence for oxidative phosphoryation in malarial parasites is not available, the presence of NADH dehydrogenase and NADH: oxidase has been demonstrated in mammalian parasites (187, 188). The role of O2 in the mammalian malarial parasite and its relationship to mitochondrial function remains unclear. It has been suggested that electron transport in plasmodia is coupled to gg'ngyg synthesis of pyrimidines and serves as a disposal for excess reducing equivalents. (74). As well, the increased glycolytic rate observed for infected erythrocytes would result in depletion of NAD+ unless it is quantitatively regenerated by the reduction of pyruvate to lactate. However, for P. knowlesi (143), under aerobic conditions lactate accounts for not more that 50% of the metabolized glucose and could not completely regenerate NAD+. Again, it has been suggested that electron transport may serve to dispose of excess reducing equivalents and serve to regenerate oxidized NAD (153). Oxygen has been postulated to act as the terminal electron acceptor. The composition of the electron transport chain has been, at best, partially described. Cytochrome oxidase ac- tivity has been identified in P. knowlesi and P. falciparum (139, 189) and histochemically localized in the mitochon- dria of P. berghei (190). Ubiquinone—8 has been con- 19 sistently identified in other mammalian species (191, 192) and analogues of this compound are inhibitory to parasite development (193, 194). The electron transport inhibitors antimycin and KCN inhibited development of P. knowlesi, P. berghei, and P. gallinaceum _i_n vitro (74). The antima- larial drugs primaquine and menoctone were found to cause mitochondrial swelling in erythrocytic stages of P. berghei (195). Recent experiments using P. falciparum have indicated that the mitochondrion may be the site of action for many antibiotics. 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Fine structural changes observed in the erythrocytic stages of Plasmodium berghei berghei following exposure to primaquine and menoctone. Annals Trop. Med. Parasitol., 64:203. CHAPTER 1 NUTRITIONAL REQUIREMENTS OF PLASMODIUM FALCIPARUM IN CULTURE. I. EXOGENOUSLY SUPPLIED DIALYZABLE COMPONENTS NECESSARY FOR CONTINUOUS GROWTH Alan A. Divo and James B. Jensen Abstract Continuous cultivation of Plasmodium falciparum presently requires the nutritionally complex medium, RPMI 1640. A basal medium of KCl, NaCl, NaZHPO4, 3)2, glucose, reduced glutathione, HEPES buffer, Ca(NO MgSO4, hypozanthine, phenol red (in RPMI 1640 concentration), and 10% (v/v) exhaustively dialyzed pooled human serum was used to determine which vitamins and amino acids had to be exogenously supplied for continuous cultivation. Supplementation of basal medium with calcium pantothenate, cystine, glutamate, glutamine, isoleucine, methionine, proline, and tyrosine was necessary for continuous growth. This semi-defined minimal medium supported continuous growth of four isolates of P. falciparum at rates slightly less than those obtained with RPMI 1640. Adding any other 36 37 vitamin or amino acid did not improve growth. Incorporation of several non-essential amino acids , particularly phenylalanine and leucine , into proteins was markedly enhanced in the minimal medium compared to RPMI 1640. 38 INTRODUCTION Continuous cultivation of P. falciparum was originally achieved with a nutritionally complex medium, RPMI 1640 (RP), supplemented with human serum (55). Although some success has been achieved in characterizing dialyzable (15, 25) and non-dialyzable (25, 33, 52) serum factors essential for the in XEEEE maintenance of this organism, little progress has been reported in the analysis of the components of RP that are necessary for continuous culture. Experiments with other Plasmodium species, mostly in XEXQI have suggested that pantothenic acid (3, 4, 6, 47, 49-54), biotin (44,48), pyridoxine (9, 32, 54), p—aminobenzoic acid (PABA) (l,2,5,l6,22,23,30,44,52,56), riboflavin (39,46,54), folic acid (16, 42, 52), and certain amino acids, particularly isoleucine and methionine (7, 36, 37, 40—45, 56), are important for survival and multiplication. The numerous attempts made to discover the nutritional require- ments of Plasmodium spp. illustrate the interest generated by this topic; however, most of these studies were done in M; the effects of vitamin depletion on the host were difficult to separate from those on the parasite, except for PABA. Studies conducted in XAEEE used parasites that could not survive in the culture system and must be considered somewhat suspect. The development of techniques for 39 continuous culture of P. falciparum (26, 55) allowed us to investigate the requirements for some of these nutrients. Exhaustive dialysis of human serum eliminates low— molecular-weight, water-soluble compounds; dialyzed serum is inadequate for culture (18, 25) unless supplemented with hypoxanthine (15). It is difficult to eliminate such compounds from intact erythrocytes, which may provide the parasite with an adequate supply of some nutrients (34, 35). Therefore, only amino acids and vitamins that must be must be exogenously supplied by serum or medium (RP) were examined; the contribution of erythrocyte nutrients present endogenously was not determined. MATERIALS AND METHODS 3TC strain of P. falciparum (27) were used in most experiments. Additional Parasites. Cultures of the FCR strains examined included FCMSUl/Sudan (Divo and Jensen, in preparation), Viet Nam Smith (VNS), and Hondurasl/CDC. Parasites were routinely maintained in candle jars according to established techniques (26). Serum and erythrocytes. Pooled human serum A+ (PHS) and pooled bovine serum (PBS) were prepared as previously described (14). Pooled human serum was dialyzed (DPHS) at 40C for 3 days against phosphate-buffered saline plus glucose (PBSG) (NaCl, 8.78 g/l; Na2HPO4, 0.68 g/liter; KH P04, 1.42 g/liter; glucose, 2.0 g/liter; pH 7.4) to a 2 40 6 (Spectrapor tubing, 6000- final serumzbuffer ratio of 1:10 8000 MW cutoff). Erythrocytes obtained from volunteers by venipuncture were stored in citrate-phosphate-dextrose (8) and used when 1-4 weeks old. Immediately prior to use, all cells were washed 3X with 100 vol PBSG. Media preparation. All reagents and chemicals were obtained from Sigma Chemical Co. Experimental media were prepared in triple glass-distilled water and always contained the following components (final concentrations, mg;liter): D-glucose, 2000: KCl, 400; NaCl, 6000; MgSO4, 3)2'4H20, 100; NaZHPO4'7H20, 1512; hypoxanthine, 4.1; glutathione, 1.0; phenol red, 5.0; and HEPES, 5940. 48.84; Ca(NO They’ were combined and used as a 10X concentrated stock solution. Most concentrations are the same as in RP (GIBCO, Grand Island, NY, Cat. #H—18); hypoxanthine and HEPES were supplements. Amino acids (L-isomers) and vitamins were prepared at concentrations 100- or 1000-fold greater than final concentrations. When added to experimental media, the final concentrations in mg/liter, equal to RP were: arginine, 200; asparagine, 50; aspartic acid, 20; cystine, 50; glutamic acid, 20; glutamine, 300; glycine, 10; histidine, 15; isoleucine, 50; leucine, 50; lysine HCl, 40; methionine, 15; phenylalanine, 15; proline, 20; serine, 30; threonine, 20; tryptophan, 5; tyrosine, 20; valine, 20; biotin, 0.2; calciunl pantothenate, 0.25; choline Cl, 3.0; folic acid, 1.0; inositol, 35.0; nicotinamide, 1.0; p-aminobenzoic acid, 41 1.0; pyridoxine HCl, 1.0; riboflavin, 0.2; thiamin HCl, 1.0; and vitamin B12 (cyanocobalamin), 0.005. Briefly, 1 liter of medium was prepared by mixing 100 ml of the 10X stock solution, 10 ml of the appropriate amino acids, 1 ml of the appropriate vitamins, and enough triple- distilled H20 to make 960 ml. The pH was adjusted to 6.65 and media were sterilized by passage through 0.45-um cellulose acetate filters (Schleicher and Schuell, Inc.). Then 40 ml of 5% (w/v) NaHCO3 was added. Final preparations also included 10% (v/v) PHS, 5 or 10% (v/v) DPHS, or 10 or 20% (v/v) PBS. Control cultures grown in RP were supplemented with 10% (v/v) PHS (RP—10) or 10% (v/v) DPHS (RP—10D) plus 4.1 mg/liter hypoxanthine. Medium containing all amino acids and vitamins was termed "reconstituted" and was supplemented with 10% (v/v) DPHS (Recon-10D). It should be noted that four Selectamine Kits (GIBCO) of two different lots failed to support parasite growth even when fully reconstituted; the reasons for this failure were not investigated. Stability of both refrigerated and frozen reconstituted medium was comparable to RP in preliminary experiments. Assessment of parasite growth. In some experiments, the incorporation of [3H]hypoxanthine (10 Ci/mmole, New England Nuclear Corp.) into parasite nucleic acids was used to monitor parasitemia in 96-well microtiter plates (Flow Laboratories, Inc.) by standard techniques (10, 13, 19, 21, 28). Each well contained 200 ul of the appropriate medium 42 and 2 m1 infected erythrocytes. Cultures were grown in a candle jar for 48 or 96 h. In 48—h experiments, [3H]hypo- xanthine (10 uCi/ml final concentration) was included in each. well for the entire incubation period. Experiments were begun with parasites in the schizont stage, synchronized (28) by a combination of sorbitol lysis (29) and gelatin flotation (24), at 0.8-1.0% parasitemia. In 96- h experiments, incubation was initiated with synchronous schizonts at 0.2% parasitemia. These cultures were grown for 48 h in the absence of [3H]hypoxanthine as before. These cultures were allowed to grow for an additional 48 h. At the end of the incubation period, cultures were harvested with a Bellco Microharvester onto fiberglass filters. Filters were dried, immersed in Formula 963 scintillation fluid (New England Nuclear Corp.), and counted in a Beckman LS 7500 liquid scintillation spectrometer. In long term experiments cultures were grown in 35 mm plastic petri dishes (Corning) in candle jars (26) and parasitemias were determined by microscopic evaluation of Giemsa-stained thin blood films. Such experiments were initiated with schizonts synchronized as above at 0.1-0.2% parasitemia. Parasites were subcultured every 96 h to parasitemias of 0.1-0.2%. Amino acid incorporation. Incorporation of [3H]amino acids (New England Nuclear Corp.) into parasite proteins was determined in 96-well microtiter plates during 48—h exposures. Cultures were prepared and harvested, and 43 radioactivity was determined as described above. All [3Hlamino acids were used at 10 uCi/ml. RESULTS The effects of omission of single amino acids or vitamins from Recon-100 medium on parasite growth for 48 and 96 h are presented in Table I. Growth was markedly reduced when no amino acids were present. Deletion of cystine, glutamate, or glutamine reduced [3thypoxanthine incorpora- ation by 40-60% at 96 h, and deletion of proline or tyrosine caused slight but significant decreases by 96 h. When all amino acids were present but all vitamins were eliminated, growth was poor. This effect was reversed by the addition of Ca2+ pantothenate; deletions of other vitamins had no effect on [3H]hypoxanthine incorporation in 96-h cultures. When pantothenate and various amino acids were added to the basal medium, continuous growth was observed only if cystine, glutamate, glutamine, isoleucine, methionine, proline, and tyrosine were added (Table II). Cysteine could replace cystine completely (data not shown). Addition of other amino acids or increased concentrations of pantothenate, glutathione, or hypoxanthine was of no benefit (data not shown). Basal medium containing pantothenate and these 7 amino acids (minimal medium) was used to culture parasites for 20 days. Comparative growth rates iJ1 RP-10, Recon-10D, and 44 .3 mm Mom mfieouflmwumm mm.o Wm one s we now mflEWuHmmumm m>.o um mucoNHnom msocounocmm nufl3 comma mums mousuasu .Azmm + cmoEV 5 mm on mmoa + m.vv¢.aa can a we on vmm + >.mmm.ma oumz emu Houucou .« couococ ma Ho>mH mo.o v A map on Houucoo ou coummeoo cofluoscou unmowwflcmfim .mcofium>uomno xflm mo some one mucomoumwu osHm> comm .Awaofluoaoc ocv Homecoo mo w mm noncommum mum mama m O~.sO HO.AHH menace new «mam OO.OHH mm.OO maflaomuaa O~.OHH OO.mOH shamans HH.OHH AO.AOH mcflumm HH.OOH OO.OOH an>mfimonfim OO.OO mO.OOH mcflaoua OO.OHH ms.ama wcnxoenusm NO.ONH OO.NO maflcmfimflscmnm «VO.OH .mO.OO mbmcmnoouamm OO.4N OO.~A mcH:OAnumz HO.¢O mm.OOH Ofiom oflomcmnoeflemua .NA.OHH .AN.OO mcflmsa mO.kO OO.mO menemcnuooflz OO.HNH Om.sO mcsoswq NO.OOH NO.m~H HebflmoeH mm.sfl 5O.ms mcflosmHOmH OO.OO OO.NHH mnmfioa .Om.mNH .4m.mm mcflanbmnm OO.OO NH.mOH chemfimnooocmso mm.mma O~.OO manosfio OH.OO me.OOH mcnfioao ON.mO mm.sm mensmbsfio mm.OOH mm.OOH cfluoflm .Om.~m .mO.me womempsfio mm.OH ms.ms manemufl> Hfia .Om.OO .ma.mm mcflbmso OOH OOH mcoz «Om.OOH «Om.OO wumuummma OO.OOH «OO.OO mcflmmummma sm.OO Oe.mO mcflflm> NO.OOH OO.mO maflcnmua «O.OO OO.OO mcnmouse mm.OH Hm.sv menom ocflem Hfla NO.~mH as.mO seasonasua . OOH . OOH 6:02 : Om a we :pomHama 2 mm g O4 aohuwfimmn :oflumnomuoocfl onwnucmxomwcmmma coflumuomuooafl maficucmxommnflmma musuflso CA s cm no mv How Esummflofimm enacOEmmHm mo opmfiomfi uammum on» we coHumuomuooca ocficpcmxommn an L mo EDchE noalcooom sown mcfiemufl> no mcwom ocflem oamcflm mo cowuoflmmc mo muoommm kw mqmumwno wlm mow 2mm H some mucomwumou o5~m> zoom .Amovwooueumuo ooom Mom mouflmmummv mEHHw cooHn :flcu cocflmumlwmeoflo mo coflpmsHm>w osmoowonofle we cocfleuouwc mmfleouflmmumm n .mme A>\>0 woa eufl3 coucoemammdw mm3 EDHcoE coco .oalmm now pmooxm .MHEopHmmuwm wH.o um muaoNHcom msocounocww nufiz comma ouo3 mucwEHuomxm o no.m H no.m mm.o H vo.v mo.o H mn.o emu + one + :H0 + Dam + who + “we I l I + oHH + ouccocuoucmm + ssficos memm HN.0 + hv.0 mH.0 + Nm.H 00.0 + No.0 Cam + 5H0 + m>o + qu + THO l I I + oumcwcuoocmm + eoflcos Hmmmm HH.0 + HN.0 0H.0 + 00.0 v0.0 + wm.0 HOE + OHH I I I + muwcocpoucmm + ssflcoe Hommm m0.0 + m0.0 00.0 + NN.0 m0.0 + mm.0 wUHOM OCHEM 0G 5 I I I + oumconuoucmm + szficoe Hommm 4 Hm.0 + ma.m NN.0 + mh.m m0.0 + 05.0 wUHUm OCHEM HHM I I I + oumcocpoocmm + ssflcoe Hommm v¢.0 H vv.m mm.0 H mm.m 50.0 H mm.0 QOHIcoowm hm.0 + mm.m 0N.0 + Hm.v 00.0 + 05.0 oalmm on mmH on em on wv mmdouw onesewufimmnmm .mcflom ocfiem wsoflum> can opmcoQDODCMQ mcflcflmucou EDHcoE Hmwmn CH EDHmmHUHMw Esflcoswmflm mo oumHOmfl UBMmUm mo suzouw .HH mqmuomno usom mom 2mm H some mucomonmwu Hones: comm .mEHflw cooHn seep cocflwumIMmEon mo qofiumcflemxo oflmoomouofie he cmcfleuoooc A: mqv ofioxo mom mwsooflmmumm CH ommouocfl some may mucomoumou osfim> comm m mm.O M HH.O 40.0 m OO.A m0.0 m mm.m NO.H w HA.O OOHIHmsfleflz OO.H + «O.HH OO.H + Os.HH sO.O + OA.O «HO.H + Os.OH Oaumm H oem UQU\mmusccom mz> :mc:m\ szum mum Esflpwz shadow .ESHUoE HMEHCHE cam calmm CH Esnmmflofimm EDHUOEmmHm wo mopmflomfl .UBm UQU\waHSUC0m Ucm .mZ> .Emcsm\abmzum mom wnu mo wGOmHHMQEOO £¥3OHD .HHH mqmdfi 50 TABLE IV. Incorporation of [3H]amino acids into proteins of the FCR3TC isolate of Plasmodium falciparum during 48-h exposures. Incorporation (cpm)b'C Amino acida Minimal-10D medium RP-lO medium Arginine (43) 14,633.0 1 258 1223.8 1 115 Asparate (34.4) 325.5 1 17 136.4 1 8 Glutamate (60) 301.3 1 17 209.1 1 35 Glycine (49) 3299.5 1 95 3112.2 1 176 Histidine (56) 5121.4 1 257 3556.6 1 132 Isoleucine (113) 7467.2 1 594 9424.7 1 391 Leucine (71) 47,072.7 1 3727 8803.8 1 377 Lysine 75) 15,213.2 1 657 2836.5 1.70 Methionine (80) 1120.1 1 142 1961.9 1 188 Phenylalanine (82) 65,595.2 1 2374 16,220.6 1 915 Proline (114) 6654.9 1 470 5119.1 1 403 Threonine (57) 2030.1 1 100 741.6 1 66 Tyrosine (45) 14,522.1 1 449 11,553.5 1 605 Valine (32) 17,711.8 1 766 7384.0 1 213 a Each amino acid was added at 10 uCi/ml; specific activities in Ci/mmol give in parentheses. b The experiment was initiated with synchronous schizonts at 0.92% parasitemia. Each value represents the mean 1 SEM of six observations. C Mean cpm (six observations) for other potential nutrients in these experiments were (minimal-10D vs. RP-lO):[3H]hypoxanthine, 16,974 vs. 24,806; [3H]guanosine (5' - [3H], 25.1 Ci/mmol, 20 uCi/ml, 1975 vs. 176; [14C1ribose (1-[14C], 53 mCi/mmol, 2 uCi/ml), 486 vs. 305; [14C1acetate (1,2—[14c1, 54.15 mCi/mmol, 2 uCi/ml), 790 vs. 641; [14C] alpha—ketoglutarate (1-[14C], 15 mCi/mmol, 2 uCi/ml, 2 uCi/ml), 328 vs. 440. .mououfioo oalmm ca mucoNHcom wmo woos mooflmmumm .mouspfino oalmm cw mpcouflcom wam mums mouflmmumm o .wousufiso oatmm CH mpcoNflnom wmo oumz mwuflmmumm w .mcofium>uomno mum now 2mm H some mucomoumou wsHm> zoom .HH ofioma an no pocflsuouoo wmfleouflwmumm a .MHEmDHmmumm wmm.o um mucoNflnom msoooucoc>m epa3 comma ouo3 mucwEHmexw one m 51 va.o + mm.0 wm.o + vv.v no.0 + mm.H mmm A>\>0 wofi + CH0 + Dam + mmo + uoE + oHH + oumcocooucmm + Esflpoe Hmmmm no.0 + 0a.0 00.0 + mm.« 0H.o + vm.a mum A>\>0 woa + woe + oHH I I I + oumcoSDODGMQ + EDHcoE Hommm no.0 + mm.o mm.o + mn.m 00.0 + 0H.H wmm ~>\>0 wow I I + oumcozuouowm + EDHooE Hmwmm 0 00.0 + m0.a 00.0 + no.0 mmm A>\>0 woa I I I + mumsocuopcmm + ESHcoE Hommm v0.0 + no.0 nN.o + mo.m m0.o + m.H mmm A>\>0 moH I I I + opwcocuoucmm + fiancee Hmwmm Ho.o + Ho.o NH.0 + 00.H 0H.o + on.o mmm A>\>0 wm I I I + oumconuoucmm + esflcoE Hmwmm mm.o + no.0 H0.o + mo.n HH.0 + mm.a oalmm on mma on 00 on we mmsouo emmfleouflmmumm mmm no mmm paw .moflom ocflsm wooflum> can .owmcosooucmm LDHB coucosofimmsm osmmoa one no nnzouo .> mamas EDOUoE Hommo CH esummwofimw Esflposmmfim mo oumHOmH 52 DISSCUSSION Although growth of "cell-free" P. lophurae was obtained in a special medium supplemented with red cell extract (49), it is currently impossible to culture P. falciparum outside of erythrocytes. The structural and metabolic functions of the erythrocyte that are critical for development and multiplication have not been well characterized. For instance, erythrocytes contain considerabe concentrations of many amino acids and vitamins (35) that may be important to the parasite. It is difficult to determine if experimental manipulations that decrease viability do so because of effects upon the parasite itself or upon erythrocytes which render them unfit hosts for survival or invasion. Thus, it is not possible to determine if nutrients identified as crucial for parasite cultivation are required for erythrocytes, parasites, or both. For instance, glutamate, glycine, and cysteine, the components of glutathione, are necessary for long-term survival of erythrocytes in culture (17). Currently, in vitro cultivation of P. falciparum requires a complex medium, such as RP, containing at least 5% (v/v) PHS, PBS, or rabbit serum (14, 26, 38). The medium contains a variety of low molecular weight components, including vitamins and amino acids, while serum supplies similar compounds which are dialyzable and others, probably proteins, which are non-dialyzable (MW 6000-8000). The 53 present studies have only examined low-molecular-weight compounds. The finding that hypoxanthine (or other purines) is the major dialyzable serum component necessary for parasite culture (15) allowed exhaustive dialysis to eliminate other serum-derived low molecular weight compounds in the culture medium. Preliminary' experiments had shown that. adequate concentrations of glucose and glutathione were necessary (data not shown). These compounds were thus present in all media at concentrations found in RP. Hypoxanthine was added to all media at 4.1 mg/liter, a concentration which is adequate to support cultures (15). Changes in electrolyte or salt concentrations were uniformly deleterious (data not shown) and so these components were always present at RP concentrations. When vitamins were deleted singly from a totally reconstituted medium, only omission of pantothenate impaired viability (Tables I, II). When amino acids were deleted in similar experiments, cystine, glutamine, glutamate, isoleucine, methionine, proline, and tyrosine were identified as necessary for continuous cultivation (Tables I, II). When these components were added to the basal medium, four strains of P. falciparum grew continuously at rates only slightly lower than those observed with RP (Fig. 1, Table III). Whether the discrepancy between growth rates is due to variability in medium formulation, to the advantages of exogenous supplies of Hb-derived amino acids, 54 or to the absence of some trace element or other compound is not clear. It is possible that some adaptation might have occurred during these experiments so that substrains of parasites able to grow in the minimal medium were selected, but careful consideration of the data presented in Fig. 1 and in Tables II and III does not reveal any evidence of this phenomenon since parasite multiplication rates were consistent in RP and the minimal medium. The importance of an exogenous source of pantothenate for Plasmodium species is well documented (3, 4, 6, 47-54). Absence of acetyl CoA inhibited the development of erythrocyte—free P. lophurae 13131119 and dietary deficiency of pantothenate markedly reduced parasitemias in birds (3, 4, 6, 50, 53). Pantothenate dependence also illustrates the complexity of the erythrocyte-parasite relationship; the host cell supplies the enzymes for the synthesis of acetyl CoA from pantothenate, which are lacking in the parasite (3,6). It is thus not surprising that pantothenate must be exogenously supplied for P. falc1parum cultivation. Other investigators, primarily using 12 1119 models, have demonstrated that dietary deficiencies of other vitamins, including p-aminobenzoic acid (1, 2, 5, 16, 22, 23, 30, 44, 52, 56), riboflavin (39, 46, 54), and bdotin (43, 48) can reduce parasitemias. Observations on the distribution of inherited deficiencies of enzymes involved in pyridoxine metabolism have led to the suggestion that this vitamin is important for human Plasmodium species (9, 55 32). During prolonged dietary deficiency, concentration of these vitamins 1J1 serum and zui tissues, including erythrocytes, drop markedly (12) to a point where parasite multiplication is prevented; however, in healthy individuals it appears likely that erythrocyte stores of these compounds are sufficient for the parasite. Extensive washing of red cells in vitamin-free buffer did not impair growth. Thus, simply depleting vitamins from culture medium may not identify some compounds that are critical for parasite success. The observation that the absence of biotin decreased the early in 31112 development of P. knowlesi in rhesus monkey erythrocytes (44) cannot be extended to P. falciparum. These observations illustrate the benefits of an 12 11119 culture system for such analyses, since studies i_n_ 11319 are plagued by difficulties in accurately determining extra- and intracellular vitamin levels in test animals and the complications of vitamin deficiency on the general health of the host. Furthermore, in XEEEQ studies with organisms such as P. knowlesi were always marred by the inability of available culture systems to maintain such species continuously. Despite these extensive observations, none of these vitamins were required for growth of .P. falciparum in culture. It is possible that vitamins could leak from uninfected erythrocytes during the 96-h incubation period, providing a secondary source for the parasites. Although there are relatively few data relating specifically to vitamin 56 pharmacodynamics in human erythrocytes, other systems provide ample evidence that vitamins such as riboflavin, nicotinamide, pyridoxine, and thiamin are quickly (if not simultaneously) metabolically activated by phosphorylation and /or adenylation upon transport (11, 31, 35, 58). Such forms are relatively impermeant and would be expected to have a low rate of efflux from the erythrocyte. Growth of P. falciparum in Waymouth's medium, which lacks PABA, has recently been reported (5). This medium contains folic acid, which may serve as a PABA source (16), and the experiments used undialyzed human serum which contains PABA. It is clear from the antimalarial effect of the sulfonamides, which are PABA antimetabolites, and dihydrofolate reductase inhibitors such as pyrimethamine, that PABA metabolism to folate is essential for Plasmodium spp. The finding that neither vitamin is required for cultivation indicates that intraerythrocytic stores of PABA are adequate for parasite success; for PABA, exogenous requirements do not reflect actual endogenous requirements in these strains. Amino acid uptake and incorporation have been studied in a variety of Plasmodium species (7, 36, 40-45, 56); these parameters are not always correlated with relative amino acid abundance in hemoglobin (43) and do not necessarily identify the amino acids that must be exogenously supplied for parasite culture. Isoleucine, methionine, glutamine, and cystine have been identified as amino acids which 57 enhance the success of short-term cultures of P. knowlesi (7, 36, 37, 42, 45, 55); glutamate, proline, and tyrosine must be added to these for continuous cultivation of P. falciparum. Short-term growth of P. falciparum was obtained when the only source of amino acids was undialyzed PHS or PBS; viable parasites were present even after 48 h. Longer cultivation, however, could only be obtained with more extensive supplementation. As with the vitamins, the contribution of erythrocyte stores of amino acids is difficult to determine in these experiments. The relationship between the incorporation of an amino acid. and requirements for continuous cultivation is not clear. Competition between labeled and unlabeled isoleucine occurred even in the minimal medium, depressing incorporation. Furthermore, it is known that several amino acids may share the same transport process, and interactions at this level may have decreased incorporation of some amino acids in RP anui 111 the minimal medium (59). There is evidence that parasite infection as well as simple cultivation procedures alters erythrocyte transport of tryptophan (20), another factor which must be considered. Leucine, lysine, phenylalanine, and arginine demon- strated the greatest difference in incorporation in minimal- 10D compared to RP-lO; however, these amino acids are not required for continuous culture and do not improve growth when added to the minimal medium. Leucine, lysine, and phenylalanine are among the most prevalent amino acids in 58 human hemoglobin (Hb) (57) and are also present in blood at nearly millimolar concentrations (57). Arginine, while somewhat less abundant in Hb, is also found in considerable concentrations in blood. These four amino acids are not distinguished by Hb abundance or blood concentrations from valine, threonine, histidine, or glycine, which do not demonstrate large differences in incorporation between minimal-100 and RP-10. Whereas isoleucine, methionine, cystine, and glutamine are present in at best very low amounts in Hb, proline, tyrosine, and glutamate are fairly abundant. Selective transport facilities may exist for any or all of these amino acids in P. falciparum; evidence from other Plasmodium species suggest that this is not the case for all (40, 50). These results have several implications. First, the minimal medium represents a: less expensive alternative to commercially' available media, which. may' be difficult to obtain in some areas. The observation that parasite viability can be maintained for at least 2 days in serum- supplemented basal medium may help to prevent culture loss when supplies of medium are temporarily exhausted or un- available. Second, the use of [3H]leucine or [3Hlphenyl— alanine 1J1 this medium enhances radiolabeling CHE parasite proteins. Third, a variety of metabolic studies are feasible in a simple medium in which levels of nutrients can be easily manipulated. Finally, this medium allows the characterization and identification of potential 59 antimalarial antimetabolites in a system free of high concentrations of target vitamins. ACKNOWLEDGMENTS We wish to acknowledge the excellent technical assistance of Rebecca Johnson and Linda Rissman. This work was supported by a grant from the UNDP/World Band/WHO Special Programme for Research and Training in Tropical Diseases and by NIH Training Grant AIO7203. This is journal article number 10865 of the Michigan Agricultural Experiment Station. 6O LITERATURE CITED 1. Anfinsen, C. B., Geiman, W. M., McKee, R. W., Ormsbee, R. A. and Ball, E. G. 1946. Studies on malarial parasites. VIII. Factors affecting the growth of Plasmodium knowlesi in vitro. J. Exp. 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J., Okoye, V. C. and Esan, G. J. F. 1978. Low erythrocytes pyridoxal kinase activity in blacks: its possible relation to falciparum malaria. Lancet, i:466-468. 33. Nivet, C., Guillotte, M. and Pereira da Silva, L. 1983. Plasmodium falciparum: one—step growth 111 a semi- defined medium and the stimulatory effect of human seric lipoproteins and lipososmes. Exp Parasitol., 545:147-151. 34. Oelschlegel, F. J. and Brewer, G. J. 1974. Parasitism and the red blood cell, in Surgenor, D. MacN., ed., The Red Blood Cell, Vol. 1, Academic Press, New York, pp. 93—146. 35. Pennell, R. B. 1974. Composition of normal human red cells, 15 Surgenor, D. MacN., ed., The Red Blood Cell, Vol. 1. Academic Press, New York, pp. 93—146. 36. Polet, H. and Conrad, M.E. 1968. Malaria: extracellular amino acid requirements for 13 vitro growth of erythrocytic forms of Plasmodium knowlesi. Proc. Soc. Exp. Biol. 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Biochem., 1:635-637. 44. Siddiqui, W. A., Schnell, J. V. and Gieman, Q.M. 1969. Nutritional requirements for in vitro cultivation of a simian malarial parasite, PlasmEEium knowlesi. Mil. Med., 134:929-938. 45. Siddiqui, W. A. and Schnell J. V. 1972. 13 vitro and 12 vivo studies with Plasmodium falciparum and Plasmodium knowlesi. Proc. Helm. Soc. Wash. (sp. issue), 39:204-210, 46. Thurnham, D. I., Oppenheimer, S. J. and Bull, R. 1983. Riboflavin status and malaria in infants in Papua New Guinea. Trans. R. Soc. Trop. Med. Hyg., 77:423-424. 47. Trager, W. 1943. Further studies on the survival and development 1g vitro of a malarial parasite. J. Exp. Med., 77:411-420. 48. ------ 1947. The relation to the course of avian ma- laria of biotin and fat-soluble material having the biolog- ical activities of biotin. J. Exp. Med., 85:663-683. 49. ------ 1950. Studies on the extracellular cultivation of an intracellular parasite (avian malaria). I. Development of the organisms in erthrocytic extracts, and the favoring effect of adenosine triphosphate. J. Exp. Med., 92:349-366. 50. ------ 1954. Coenzyme A and the malarial parasite Plasmodium lophurae. J. Protozool., 1:231-237. 64 51. ------ 1957. The nutrition of an intracellular para- site (avian malaria). Acta Tropica, 14:289-301. 52. ------ 1958. Folinic acid and non-dialyzable material in the nutrition of malaria parasites. J. Exp. Med., 108:753-772. 53. ------ 1966.Coenzyme A and the antimalarial action 11 vitro of anti-pantothenates against Plasmodium lophurae, P. costneyi, and P. falciparum. Ann. N. Y. Acad. Sci., 28:1094- 1108. 54. —————— 1977. Cofactors and vitamins in the metabolism of malarial parasites. Factors other than folates. Bull. W.H.O., 55:285-289. 55. Trager, W. and Jensen, J. B. 1976. Human malaria para- sites in continuous culture. Science, 193:673-675. 56. Trigg, P. I. 1982. Cultivation techniques for the erythrocytic stages of malaria parasites. Bull. W.H.O., 47:357-373. 57. White, A., Handler, P. and Snith, E. L. 1968. Prin— ciples of Biochemistry. 4th ed. McGraw-Hill, New York. 58. Wolfe, S. J. 1957. The effect of administration of thiamine analogues (oxythiamine and pyrithiamine) on rate erythrocyte metabolism. J. Biol. Chem., 229:801-808. 59. Young, J. D., Jones, S. E. M. and Ellory, J.C. 1980. Amino acid transport in human and sheep erythrocytes. Proc. R. Soc. London B., 209:355-375. CHAPTER 2 OXYGEN- AND TIME-DEPENDENT EFFECTS OF ANTIBIOTICS AND SELECTED MITOCHONDRIAL INHIBITORS ON PLASMODIUM FALCIPARUM IN CULTURE Alan A. Divo and James B. Jensen Abstract Several antibiotics which inhibit protein synthesis on 708 ribosomes, including clindamycin, pirlimycin, 4'-pentyl- N-demethyl clindamycin, four tetracyclines, Chloramphenicol, thiamphenicol, and erythromycinq had. antimalarial effects against Plasmodium falciparum in culture which were greatly influenced by the duration of drug exposure and by oxygen tension. In 96-h incubations, potency was increased by a factor of up to 106 over the first 48 h period and by a factor of up to 104 in 15% 02 versus 1% 02. Two aminoglycosides, kanamycin and tobramycin, had no antimalarial activity; IRifampin and nalidixic acid, which inhibit nucleic acid synthesis, were not similar to the 705 inhibitors. The mitochondrial inhibitors Janus Green, 65 66 rhodamine 123, antimycin A1, and 8-methylamino-B-desmethyl riboflavin had activities which were influenced by duration of exposure and oxygen tension. Quinoline-containing antimalarial agents, ionophores, and other antimalarial drugs were influenced to a minor extent by the duration of exposure but were not affected by oxygen tension. These data can best be explained by the hypothesis that 'antimalarial 7OS ribosome-specific protein synthesis inhibitors are toxic to the parasites by acting on the mitochondrion. 67 INTRODUCTION The increasing spread of chloroquine-resistant strains of Plasmodium falciparum (30, 55) has stimulated considerable effort toward the identification and characterization of new antimalarial drugs. Except for antifolates, most of this effort has been directed toward drugs which are modifications of the quinoline-containing antimalarial agents quinine and chloroquine (29, 38, 47), which may not escape the problems of resistance that currently plague the prototypes. Antimalarial activity is present in some antibiotics, many of which are active against P. falciparum 1n vitro at relevant pharmacological concentrations (16). Rifampin (2), Chloramphenicol (12, 39), and erythromycin (53) have demonstrable antimalarial effects in animal models, whereas tetracyclines (11, 13, 32, 36, 54) and clindamycin (7, 10, 26, 37) have been shown to be effective against P. falciparum in humans. Although no data are available on the mechanism(s) of antimalarial action of these drugs, it has been suggested that they act on parasite mitochondria which, like all eucaryotic mitochondria, probably contain 708 ribosomes (16). Recently, we have shown that P. falciparum contains a functional mitochondrion which, by virtue of its ability to concentrate rhodamine 123, actively maintains a high transmembrane potential (A. A. Divo, Th (3. Geary, H. 68 Ginsburg, and J. B. Jensen, J. Protozool., in press). However, since no information has yet been obtained on the metabolic role (if any) of the organelle in the erythrocytic stages of this parasite (45), it is difficult to directly test the hypothesis that antimalarial antibiotics act at the mitochondrial level. It has been shown that the antimalarial potency of some antibiotics is influenced by the duration of exposure in culture (16; D. J. Krogstad, submitted for publication) and, for clindamycin, by oxygen tension (Krogstad, Submitted for publication). We indirectly tested the mitochondrial toxin hypothesis by using these two variables to compare the effects of various antibiotics, mitochondrial inhibitors, and other drugs on P. falciparum in vitro. MATERIALS AND METHODS Stock cultures were maintained in candle jars (19, 49). In most experiments, the FCR3TC isolate (18) was used, although the Viet Nam Smith, FCMSUl/Sudan (manuscript in preparation), and FCR8 (28) strains were tested to control for strain differences in sensitivity. Experiments were conducted in 96-well microtiter plates (Linbro) as describe previously (15); [3thypoxanthine (10 Ci/mmol; NeW' England. Nuclear Corp., Boston, Mass.) incorporation was used as a measure of drug effects (14). Each well contained 2 ul of infected erythrocytes, 200 ul of 69 RPMI 1640 medium (GIBCO Ieboratories, Grand Island, N.Y.) supplemented. with IHEPES (N-2-hydroxy-ethylpiperazine-N'-2- ehtanesulfonic acid) (Sigma Chemical Co., St. Louis, Mo.), sodium bicarbonate, and 10% pooled human serum (RP-10), and various drug concentrations. Drug exposure occurred for 48 or 96 h. In 48-h experiments, initial parasitemias were 1 to 2% schizonts synchronized as described previously (56) by a combination of sorbitol lysis (22) and gelatin flotation (17). Each well received 2 uCi of [3H1hypoxanthine. After 48 h, the cells were harvested onto glass fiber filters with a Bellco cell harvester. The filters were dried, added to scintillation fluid, and counted with a Beckman LS7500 scintillation spectrometer (15). In 96-h experiments, parasitemias were initially 0.2 to 0.3% schizonts. After 48 h, the supernatant was carefully aspirated from each well and replaced with medium containing identical drug concentrations and 10 uCi of [3thypoxanthine per ml. After an additional 48 h, the cells were harvested as described above. 8-ethylamino-8-desmethyl riboflavin was tested exactly as described previously, except RPMI 1640 medium was replaced with a semi-defined minimal medium which lacks riboflavin (A. A. Divo, T. G. Geary, N. L. Davis, and J. B. Jensen, J. Protozool., in press). Experiments were conducted in a tissue culture chamber (Billups-Rotherburg, Inc.) gassed with a mixture of 1% O -3% 2 CO -96% N (low oxygen) or in candle jars. The mixture in 2 2 theses containers has been measured to be approximately 15% 70 O 2% C02, and 84% N2 (43). Gas phases were changed 2: daily. All drugs were initially dissolved in either 100% ethanol or in triple-distilled water at concentrations ranging from 10"1 to 10-3 M. These solutions were sterilized by filtration through 0.45-uM (pore size) membrane filters (Schleicher anul Schuell, Inc., Keene, N. H.) when necessary. All data points represent means of 3 to 12 experimental observations; standard errors were 110 % of the mean. When potencies are compared, values for 50% inhibitory concentrations (ICSOS) are defined as the drug concentration resulting in a 50% decrease in [3H]hypoxanthine incorporation compared with drug-free controls; values were obtained by simple graphic extrapolation and, as such, are only estimates. Drugs used and sources were as follows: tetracycline, oxytetracycline, minocycline, erythromycin, Chloramphenicol, thiamphenicol, kanamycin, tobramycin, nalidixic acid, Janus Green, antimycin A cycloheximide, anisomycin, aminopterin, 1, valinomycin, gramicidin, nigericin, monensin, tetraethyl- thiuram disulfide, actinomycin D, chloroquine, and quinine from Sigma; rifampin from Boehringer-Mannheim Biochemicals, Indianapolis, Ind.; halofuginone from Roussel Laboratories, Ltd., Middlesex, England; and rhodamine 123 from Eastman Kodak Co., Pochester, N.Y. The following were generous gifts: clindamycin, pirlimycin, and 4'-pentyl-N-demethyl 71 clindamycin from S. F012 and R. Westerman, The Upjohn Co., Kalamazoo, Mich.; 8-methylamino-8-desmethyl riboflavin from E. F. Rogers, Merck Sharp and Dohme, Rahway, N.J.; mefloquine from the Walter Reed Army Institute of Research, Washington, D.C.; amodiaquine from L. Werbel, Parke- Davis/Werner Lambert, Inc., Ann Arbor, Mich., and desethylchloroquine from Stirling-Winthrop, Inc., New York, N.Y. RESULTS Antibiotics that inhibit protein synthesis on 70S ribosomes, except for the aminoglycosides tobramycin and kanamycin, which were essentially inactive, showed marked dependence on exposure time and O2 tension (Fig. 1; Table 1). The most potent were clindamycin, pirlimycin, and 4'pentyl-N-demethyl clindamycin; at high 02, ICSOS at 96 h were 5.1 x 10_9, 2.2 x 10—9’ and 3.3 x 10"11 M, respec- tively. These values are between 104 and 106 times lower than those observed for the same drugs at 48 h in either high or low oxygen. The shape of the concentration—response curves for these drugs was extremely sensitive to O2 tension at 96 h, although curves were identical in both atmospheres at 48 h. Similar results were obtained for erythromycin, four tetracyclines, Chloramphenicol, and thiamphenicol (Fig. 2; Table 1). IC 5 at 96 h in high oxygen were generally about 50 s A i nu. 72 Fig. 1. Concentration response curves describing the inhibition of P. falciparum by clindamycin and related derivatives. Data were obtained with the FCR isolate. ----- , Cultures exposed for 48h; , exposggcfor 96 h. Solid circles, experiments in candle jars (high oxygen); open circles, experiments done at 1% O . Vertical bars represent standard errors and are drawn in the clindamycin figure for illustrative purposes; in every case, these values were _<_ 10% of the mean. Each point represents the mean of 3 to 12 obsegvations. Data are presented as the percent reduction in [ thypoxanthine incorporation compared with drug-drug controls. 73 z_o>22Ihm2wo -zéfizua .... IOO‘ 50- I00 . 0 5 20.5912. IOO‘ 50‘ (M) CONCENTRATION 74 .ocHwHDch Emuzflcufinnuwmuuoh .Qams I .cwuwou :oHumuucoocoo o uwwcoH: ecu . 0H on some new mo Houuo oumccmum + :oHanHccH unwound cows mum mmsHm> I «I . . . . .cme on» a mo onv one: muouum oumocmum .ommo >Ho>o cH .coHuw>uomno NH cu M NO memos mum mosHm> m :H>mHuonHu Hanumewmo OHIOH x O.m OHuOH x O.H OIOH x O.~ OH x O.v IOIocHEmHsnuszO nIOH x H.m n.OH x H.O nuOH x O.q OH x O.m ocHsvouoHcoHszuwmoO O.OH x 0.0 O.OH x ~.O O-OH x O.H OH x ~.~ ocHsuonwz OIOH x H.O OuOH x m.m O..OH x O.H OH x n.~ mcHsuchoe< nIOH x O.H O..OH x n.v nIOH x O.H OH x n.H ochHzo OIOH x O.m O.OH x «.4 nIOH x O.H OH x O.H ocHsuouoHno O.OH x O.O nsOH x O.H O.OH x m.m OH x m.v cHuwuaocHe< O.OH x O.H OuOH x O.H O..OH x O.m OuOH x O.m ocochsuonm nIOH x O.O nIOH x ~.m O..OH x m.~ O:OH x m.~ oases HHIOH x O.H HHIOH x O.H HHIOH x ~.m HHIOH x O.m :HuHmeHz OHIOH x n.H OHIOH x H.H OHIOH x O.H OHuOH x ~.m chcocoz HHIOH x O.~ HHIOH x O.H OHIOH x H.H HHIOH x ~.O :HoHoHeouo OIOH x O.H OuOH x m.H OuOH x O.m OuOH x ~.m :HonsocHHu> OHIOH x O.O OHIOH x H.m O.OH x H.H OHIOH x H.O a :HossocHuoa muOH x O.H TOH x O.H TOH x ~.m TOH x H.m oHoc onHuHHuz OH x O.H OH x O.m OH x v.~ OH x O.H cheuuHm bl hl bl hl OO.H H OO.HH OO.H H NO.mH H~.O H H~.H -.O h Hm.~ ncHoseuunoa OO.~ + Om.vm m~.m . Om.Om OO.H . mm.O~ Hn.H + On.OH :Hossmcux mIOH x O.~ OuOH x O.~ TOH x O.H muOH x H.O HooHcocmeana HIOH x O.m OuOH x O.~ OnOH x O.m OnOH x O.m wcHHosuocH: O.OH x n.n nsOH x O.k muOH x O.H OIOH x O.~ ucHHosouHuuuuoHno OIOH x O.~ nIOH x m.m OIOH x O.H m.OH x ~.m wcHHusouHuuusxo No 30H No :OH: No 30H No :mHz mayo r OO 2 we "econcwu No new wcw3oHHom um A20 omUH .wumHomH oemmom. esummHonu .M mo ousufiso :H mucomm HafiumHmsfiucm mo nocouom :o :omnxo can oEHu mo cocoSHMCH .H mqmo_zomx._.>mm HOG-zmzag‘dmOJIo IOO" 50“ I00 50'1 ZO_._._m_Iz_ .x. IOO 50‘ (M) CONCENTRATION 77 100 times lower than those observed at 48 h. 02 tension did not affect potency or efficacy at 48 h but had profound influence in 96-h incubations. The effects of anisomycin and cycloheximide, which inhibit protein synthesis on 808 ribosomes, were identical in high and low 0 and were not increased by prolonging 2 exposure (Fig. 3). Antibiotics which inhibit nucleic acid synthesis, including actinomycin D, nalidixic acid, and rifampin, did not resemble the 708 inhibitors (Table 1). Responses to actinomycin D and rifampin were unaffected by O tension or incubation time; although nalidixic acid was 2 more potent at 96 h, it had greater activity in low 02. Several compounds which can act as mitochondrial inhibitors had antimalarial effects which were time dependent to a variable degree. The influence of O2 tension was variable and never as pronounced as for the 705 inhibitors (Fig. 4). Rhodamine 123, like nalidixic acid, was somewhat more potent in low oxygen. Janus Green and 8- methylamino-8-desmethyl riboflavin were somewhat more potent in high oxygen. The effect of antimycin Al was not affected by oxygen; however, the effects were reversed to some extent, particularly at low antimycin A concentrations, by 1 ascorbate (data not shown). A ‘variety' of other drugs, including the ionophores valinomycin, gramicidin, monensin, and nigericin; the quinoline-containing antimalarial agents quinine, chloroquine, mefloquine, amodiaquine, and desethylchloro- 78 Fig. 3. Concentrations-response curves describing the effects of the 808 ribosome inhibitors anisomycin and cycloheximide on P. falciparum in culture. Data are presented as described in the legend to Fig. 1. 79 3 I - I I6 165 .6 CONCENTRATION I67 U '- Ii? I001 80‘ NOLLIBIHNI 9: (M) 80 Fig. 4. Concentration-response curves Idescribing the effects of the mitochondrial inhibitors Janus Green, rhodamine 123, and antimycin A on P. falciparum in culture. Data are presented as described in the legend to Fig. 1. INHIBITION % I007 50‘ I23 RHODAMINE 81 I00 50‘ 1.. g 9 z E < l00 50" GREEN 8‘ CONCENTRATION (M) 82 quine; and miscellaneous compounds with antimalarial activ- ity, including halofuginone, tetraethylthiuram disulfide, and aminopterin showed only minor increases in potency in prolonged incubations and were not affected by 02 tension (Table 1). The effects of the antibiotics and mitochondrial inhibitors were essentially identical in the FCR Viet 3TC' Nam Smith, FCMSUl/Sudan, and FCR8 strains of P. falciparum (data not shown). DISCUSSION Malaria parasites are sensitive to O2 tension (43); in our experiments, P. falciparum grown in 1% O2 incorporated about 30% more [3H]hypoxanthine than those grown in candle jars (15% 02)’ These organisms are extremely susceptible to oxidant stress (3). This sensitivity has been invoked to explain the antimalarial effects of certain chelators which may inhibit parasite or red cell enzymes involved in detoxification of oxygen radicals (41). This finding is consistent with observations that buthionine sulfoximine, an inhibitor of glutathione synthetase, has antimalarial activity in P. falciparum cultures (manuscript in preparation). In both cases, toxicity was related to O2 tension. Oxygen dependence has been demonstrated for the antimalarial effects of the imidazoles. Potency was 83 2 the O2 tension was increased from 0.3 to 18% (31). These drugs inhibit fungal increased by roughly 10-fold when the O sterol synthesis at concentrations at least 100-fold lower than those which are antimalarial (about 5 nM versus approximately 1 uM); at the higher concentrations, these drugs affect membranes and inhibit membrane-bound oxidases (51). Significantly, ndtochondrial function is; inhibited (51). Recently, the antimalarial activity of clindamycin was reported to be 0 dependent (Krogstad, submitted for 2 publication). Our findings extend this observation to a variety of other antibiotics whose only common mechanism of action is inhibition of protein synthesis on 708 ribosomes (35). Some of these drugs have been shown to have antimalarial effects 11’3132 and 12'31119 (16 and references therein) and included clindamycin, pirlimycin (6), 4'- pentyl-N-demethyl clindamycin (24, 25, 33, 34, 39), tetracycline, minocycline, oxytetracycline, Chloramphenicol, thiamphenicol, and erythromycin. The influence of O was 2 generally not evident at 48 h but was profound at 96 h. IC 5 were up to 106 times lower for 4'-pentyl-M-demethyl 50 clindamycin in high 0 The unusual pattern seen in the 2. concentration-response curves of the clindamycin derivatives at 96 h in low 0 as previously reported (Krogstad, 2' submitted for publication), was not evident for the other drugs. Pirlimycin has not been shown to have antimalarial effects before. However, it presents no advantage over 84 clindamycin in potency. On the other hand, 4'-pentyl-N- demethyl clindamycin has been shown to be more potent than clindamycin in several animal models (24, 25, 33, 34, 39) and was recommended as more promising than clindamycin (44). This compound is more potent than clindamycin in high oxygen by a factor of nearly 100. It may be that slight modifications of other antibiotics could generate a variety- of potent antimalarial agents. Of the tetracyclines, minocycline was the most potent. It is also more potent than other tetracyclines as an antibacterial agent (40) and is apparently more potent than tetracycline, oxytetracycline, and chlortetracycline against P. falciparum (13, 36, 54) and P. berghei (21, 50) in vivo. Enhanced potency may be due to the increased lipophilicity of minocycline, facilitating its entry into cells (40). Against P. falciparum, chlortetracycline was the least acive; however, in P. berghei, chlortetracycline has been found to be about twice as potent as oxytetracycline (48). Chloramphenicol is considered to be too toxic for routine use (40), and this drug has no advantages over other antibiotics for the treatment of malaria. It is interesting that thiamphenicol, a derivative which shares the antibacterial effects but not the toxicity of Chloramphen- icol (4), is somewhat more potent as an antimalarial agent 13 21119. The aminoglycosides tobramycin and kanamycin, which also inhibit protein synthesis on 708 ribosomes (35), were essentially inactive, similar to gentamycin and 85 streptomycin (16). Whether this lack of effect is due to inadequate uptake or represents a true ribosomal resistance is not known. Oxygen tension also influenced the activity of other compounds. Nalidixic acid, which inhibits mitochondrial and procaryotic DNA gyrase (8), was somewhat more potent (at 96 h) in low oxygen. A similar effect was observed for the mitochondrial-specific fluorescent dye rhodamine 123 (9, 20), which is under investigation as an anticancer drug because of the markedly enhanced killing effect observed in transformed cells (5, 23). Rhodamine 123 is believed to function. as a mitochondrial inhibitor (27) since it is highly concentrated in these organelles (9, 20). This drug is effective 11 vitro against P. falciparum at concentrations even lower than those which are toxic to normal mammalian cells (23). Another mitochondrial—specific dye, Janus Green (50), was found to inhibit P. falciparum with extraordinary potency; these effects were enhanced in high oxygen (IC at 50 96 h, approximately 10.12 In. The antimalarial riboflavin antagonist 8-methylamino-8-desmethyl riblflavin (T.G. Geary, A. A. Divo, and J. B. Jensen, J. Protozool., in press) also showed increased potency at 96 h in the high-oxygen atmosphere. Riboflavin functions as a cofactor in many enzymes which may be presumed to be important to malaria parasites, such as glutathione reductase or orotic acid dehydrogenase; orotic acid could partially reverse the 86 toxicity of riboflavin antagonists in culture (manuscript in preparation). Neither of these compounds had concentration- response curves which resembled those of the 708 inhibitors. That of Janus Green in particular was extremely gradual, resembling the electron transport inhibitor anti- mycin A 1 (50). Antimycin A was unaffected by oxygen tension but was 1 partially reversed by ascorbate; this observation adds credence to the hypothesis that antimycin A acts on 1 parasite mitochondria since, in other cells, ascorbate can partially reverse the inhibition of electron transport by directly reducing cytochrome b, bypassing the drug blockade (50). The specificity of these effects can be seen by the lack of influence of time and oxygen on the potency of a wide variety of other antimalarial agents. These drugs include the nucleic acid synthesis inhibitors actinomycin D and rifampin (35); the ionophores valinomycin, gramicidin, monensin, and :nigericin (Geary' et al., in jpress); halofuginone (15) ; aminopterin (17) ; tetraethylthiuram disulfide (42) and a variety of quinoline-containing antimalararial agents, including desethylchloroquine (1). The ionophores were extremely potent antimalarial agents, with ICSOS as low as 10-11 IL It would be interesting to determine the biochemical basis for this exceptional potency. Most importantly, the effects of the 8OS-specific protein synthesis inhibitors cycloheximide and anisomycin were not affected by oxygen or duration of exposure. 87 The inhibition of parasite growth by the 7OS-specific drugs cannot be explained by actions on the 808 ribosome. This is a critical distinction, given the observation that 808 ribosomes of coccidia (closely related to Plasmodium spp.) have some characteristics of 708 ribosomes, including sensitivity to some antibiotics (52). The complete dissim- ilarity in oxygen and time dependence between 805- and 708- specific antibiotics clearly demonstrates that the cytoplasmic ribosomes of malaria parasites are not the targets of the 708 inhibitors. This distinction extends to the tetracyclines as well, which are known to inhibit protein synthesis on both 808 and 708 ribosomes (35). Selective toxicity to bacteria is achieved because these drugs penetrate most eucaryotic cells poorly (35). However, their antimalarial potency, which is at least as great 12 211112 as their antibacterial potency (16), indicates that these drugs do enter P. falciparum. It is reasonable to expect that cytoplasmic 8OS ribosomes would be affected so that these drugs should resemble anisomycin and cyclo- heximide. Instead, they had patterns of activity identical to those of the other 7OS-specific drugs. One possible explanation of this phenomenon could be selective localiza- tion. Tetracyclines are chelators with high affinity for calcium (35), a cation known to be accumulated by P. falciparum (46). It is reasonable to assume that the organelle primarily responsible for this accumulation is as in other cells, the mitochondrion (50). Since Ca2+- 88 tetracycline complexes cannot freely pass membranes (40), this might result in the accumulation and trapping of tetracycline in the parasite mitochondrion. Recent evidence from this laboratory demonstrates that P. falciparum possesses a single mitochondrion which under- goes a complex pattern of growth, development, and replica- tion during erythrocytic schizogony (Divo et al., in press). This process was visualized with the fluorescent dye rhodamine 123, which accumulates specifically in metabolic- ally active mitochondria (9, 20). The presence of a func- tional mitochondrion has been demonstrated by the ability of a variety of mitochondrial toxins to inhibit rhodamine 123 accumulation (Divo et al., in press) and to kill the para— sites (H. Ginsburg et al., unpublished observations). The data presented here demonstrating the extraordinary antimalarial toxicity of Janus Green, rhodamine 123, anti— mycin A and 8—methylamino-8—desmethy1 riboflavin further 1’ illustrate this point. Our experiments were initiated with parasites synchronized to the schizont stage. In concentrations of 705 inhibitors which were not deleterious at 48 h, parasites appeared to be morphologically normal (16). Continuation of 7 M clindamycin) exposure to such concentrations (e.g., 10— led to complete inhibition. The progeny of parasites which developed in the first 48-h period were much more sensitive to the antibiotics than the parent. We would like to term this phenomenon the "second—cycle effect". Although we do 89 not yet understand the pharmacological basis for this effect, we propose that it is based on antimitochondrial actions. This hypothesis is currently being tested. This proposal is somewhat limited by the lack of simi- larity of the parasite response to the 708 inhibitors and to rifampin and nalidixic acid, both of which can inhibit nucleic acid synthesis in mitochondria (8, 35). Both could be expected to inhibit mitochondrial growth and development as proposed for the 708 inhibitors, but their effects are quite different. We have as yet no data to explain this discrepancy. Our proposal does not explain the mechanism(s) by which oxygen increases the toxicity of these drugs for malaria parasites. Much further work will be required to fully characterize oxygen metabolism in these organelles. Nonetheless, given the facts that the 708 inhibitors are very much alike in their patterns of parasite inhibition and that the only common mechanism of action of this structurally disparate group of drugs is inhibition of protein synthesis on 708 ribsomes, the proposal that they act as antimalarial agents by inhibiting mitochondrial protein synthesis is reasonable. With regard to the potential chemotherapeutic value of the antibiotics, several points should be made. Rifampin is an effective antimalarial agent 13 31119 at concentrations which are at or below those achieved during chemotherapy of tuberculosis (16), appears to work faster than the other drugs, and is not affected by oxygen. Although this drug is 90 expensive and not without toxicity, it may be of use as an alternative for the treatment of multiply drug-resistant falciparum malaria. Tetracyclines and clindamycin have been used in combination with other drugs for the treatment of falciparum malaria (13, 26, 54). Tetracyclines, particularly doxycyc- line, are also effective when used alone for the treatment of malaria (11, 32, 36 ).. Clindamycin, which is the most potent antimalarial antibiotic currently available, has demonstrated good clinical results when given alone for the treatment of malaria (7, 10, 37), and further clinical trials are under way. Although this drug clearly does not act rapidly enough for use in cmitically ill patients, it may be of use in treating some drug-resistant infections. It is important that antibiotics be reserved for infections which. cannot. be controlled. by' other antimalarial agents since routine use may generate resistant bacterial strains. The demonstration that these organisms contain functional mitochondria which are vulnerable to selective chemotherapy opens a new area for investigation. Antimitochondrial compounds may’ provide a :new source of antimalarial agents, and characterization of the role of mitochondrial metabolism in the erythrocytic stages of P. falciparum may identify other metabolic pathyways which are targets for chemotherapy. 91 ACKNOWLEDGMENT This study was supported in part by a grant from the World Bank/UNDP/WHO Special Programme for Research and Training in Tropical Diseases. LITERATURE CITED 1. Aderounmu, A.F., and. L. Flechenstein. 1983. Pharma- cokinetics of chloroquine diphosphate in the dog. J. Pharmacol. Exp. Ther. 226:633-639. 2. Alger, M. E., D. T. Spira, and P. H. Silverman. 1976. Inhibition of rodent malaria in mice by rifampicin. Nature (London) 227:381-382. 3. Allison, A. C., and E. M. Eugui. 1982. A radical inter— pretation of immunity to malarial parasites. Lancet ii:1431— 1433. 4. Baumelou, E., and Y. Najean. 1982. Why still prescribe Chloramphenicol in 1983? Blut 47:317-320. 5. Bernal, S. D., T. J. Lampidis, R. M. McIsaac, and L. B. 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The pmokaryotic characteristics of Eimeriatenella ribosomes. Comp. Biochem. Physiol. 61B:571- 579. 53. Warhurst, D. C., B. L. Robinson, and W. Peters. 1976. The chemotherapy of rodent malaria. XXIV. The blood schiz- onticidal action of erythromycin upon Plasmodium berghei. Ann. Trop. Med. Parasitol. 70:253-285. 54. Willerson, D., Jr., K.H. Rieckmann, P.E. Carson, and H. Frischer. 1972. Effects of minocycline against chloroquine- resistant falciparum malaria. Am. J. Trop. Med. Hyg. 21:857- 863. 55. Wyler, D. J. 1983. Malaria-resurgence, resistance, and research. N. Engl. J. Med. 308:875-878. 56. Yayon, A., J. A. Vande Waa, M. Yayon, T. G. Geary, and J. B. Jensen. 1983. Stage-dependent effects of chloroquine on Plasmodium flaciparum in vitro. J. Prtozool., 30:642-647. CHAPTER 3 THE MITOCHONDRION OF PLASMODIUM FALCIPARUM VISUALIZED BY RHODAMINE 123 FLUORESCENCE Alan A. Divo and James B. Jensen Abstract Rhodamine 123 (Rh123) has been used to probe the functional status of the mitochondrion present within the asexual, intraerythrocytic stages of the malarial parasite Plasmodium falciparum. This cationic fluorescent dye accumulates specifically in negatively charged cellular compartments, such as mitochondria. Using epifluorescence microscopy the development of what appears to be a single mitochondrion has been followed through the intraerythrocytic cycle. Mitochondrial development progress- es from a fine thread-like organelle that becomes longer and eventually branched. Each daughter merozoite receives a branch or piece of the parent organelle. Cytoplasmic Rh123 accumulation was also observed, indicating that there exists a transmembrane potential across the outer plasma and parasitophorous vacuolar membranes of the parasite. The 96 97 effects of uncouplers (protonophores), ionophores, and inhibitors were examined by monitoring Rh123 accumulation and. retention. Our results demonstrate that the mitochondrion of P. falciparum actively maintains a high transmembrane potential, the function of which is as yet undefined. INTRODUCTION Intraerythrocytic stages of mammalian malarial parasites are considered to use primarily the Embden- Meyerhof pathway for energy transduction (29). Considerable variation in mitochondrial biochemistry exists between Plasmodium species (29). The acristate morphology (1, 20) and the apparent lack of most TCA cycle enzymes (4, 29) have left the classical function of the mitochondrion in P. falciparum in doubt. Recent experiments using this parasite indicate that mitochondria may be the site of action for many antibiotics (6, 9). This Plasmodium species has been shown to be an obligate microaerophile (28) having malate dehydrogenase (35) and cytochrome oxidase (27) activity. In the present study the functional status of the mitochondrion in P. falcipaum was probed using the vital dye rhodamine 123 (Rh123). This cationic fluorescent dye accumulates specifically in negatively charged subcellular compartments, such. as active mitochondria (7, 15, 16). Using epifluorescence microscopy, Rh123 has been used to localize 98 what appears to be a single mitochondrion in P. falciparum and to follow its development over the intraerythrocytic cycle. To confirm that the observed accumulation of Rh123 was dependent upon a mitochondrial transmembrane potential, a variety of agents known to disrupt the metabolism of mitochondria were examined. The uncouplers, 2,4- dinitrophenol (DNP; 24) and carbonylcyanide m-chlorophenyl- hydrazone (CCCP; 12, 36), the ionophores, valinomycin and nigericin (25), the ATPase inhibitors, N,N'- dicyclohexylcarbodiimide (DCCD; 17), oligomycin and querce- tin (21), and the electron transport inhibitors, antimycin Al (3), n-heptquuinoline N-oxide (HQNO), rotenone, NaN3, and KCN (31) were all found to have an effect on Rh123 retention or accumulation. MATERIALS AND METHODS Parasites. In all experiments the Viet Nam Smith strain of P. falciparum were maintained in candle jars according to established methods (8, 14). Parasite suspensions were synchronized and concentrated by a combination of sorbitol lysis (19) and gelatin flotation(13). Rh123 staining. An Rh123 solution was prepared at 1.0 mg/ml in 3x glass-distilled H20 and stored at -2o°c. The appropriate concentration of Rh123 was determined by serially diluting the 1.0 mg/ml stock solution into RPMI 1640 medium sans serum (RP) and treating parasite 99 suspensions for 30 min at 38°C. In all cases, Rh123 staining' was carried out at a 1% hematocrit ‘with high parasitemias. After staining, Rh123 was removed by centrifuging the parasitized erythrocytes, removing the supernatant, and resuspending the pellet in 100 volumes of RPMI 1640 supplemented with 5% human serum (RPS). The cell suspension was returned to 38°C and periodically a small sample was centrifuged and a wet mount prepared from the pellet and observed using epifluorescence microscopy. Treatment with mitochondrial inhibitors. The inhibitors carbonylcyanide m-chlorophenylhydrazone, CCCP; N,N‘-dicyclo- hexylcarbodiimide (DCCD), and antimycin Al were dissolved in 95% ethanol and diluted with RP to the final concentration. For parasites receiving a 24-h pretreatment with DCCD, RPS was used. Drug concentration and incubation times for these compounds are listed in Table I. Other inhibitors were prepared similarly; prestained parasites were incubated for 30 min in the presence of each compound (see results). The retention of dye within both the mitochondrion and cytoplasm of the parasite was followed over time. Epifluorescence microscopy. Cells stained with Rh123 were observed and photographed using a Nikon Optiphot microscope with epifluorescence illumination and fluorescein interference filters. Photographs were taken using Kodak TRIX Pan film; exposure times varied between 9-15 sec. Reagents. Rhodamine 123 (laser grade) was obtained from Eastman Kodak (Rochester, NY) and RPMI 1640 from GIBCO 100 (Grand Island, NY). All other reagents were obtained from Sigma Chemical Co. (St. Louis, MO). RESULTS In suspensions of parasitized erythrocytes stained with Rh123 at 0.1 ug/ml, mitochondria were readily visualized in all parasite stages except the young ring forms; however, in the latter parasite stages organelle fluorescence could be seen after treatment to lyse the host cell with low-titer hemolytic serunu When Rh123 was used. at higher concentrations, or if microscopic observation was performed immediately after incubation with the dye, both the parasite cytoplasm and the mitochondrion fluoresced brightly. If the parasites were incubated for 30 min in fresh medium, however, the dye would diffuse out of the cytoplasm much more rapidly than from the ndtochondrion, leaving distinct mitochondrial fluorescence (Fig. 1). Photomicrographs could be taken for up to 3 h after Rh123 staining. Figure 1 illustrates the sequential development of the mitochondrion over the course of the intraerythrocytic cycle. Only a single mitochondrion was found per cell. Figure 1 (a-e) illustrates parasite mitochondrial development from the ring to trophozoite stage. For ring stage parasites, before Rh123 staining, the parasitized erythrocytes were pretreated with hemolytic serum. Figure 1a represents an 8-14 h ring stage parasite whose morpholgy, 101 Fig. 11. Mitochondria of .P. falciparum stained. with rhodamine 123. Sequential development over the erythrocytic cycle; a, 8-14 h ring stage parasite; b-e, 16-32 h trophozoites; f-k, 28-38 h schizonts; l-m, 36-44 h schizonts; n-q, 40-46 h segmenting schizonts; r-s, segmented schizonts containing' individual merozoites; and ta. CCCP- treated schizonts. Plates (g-i) and (l) are multiply infected. A wet mount preparation could be photographed (Tri-X, ASA 400) 13) to 3 li after staining. All photomicrographs magnified X3200. 102 103 as illustrated by the photograph, is somewhat exaggerated since 12 situ the ring stage mitochondrion has a very fine thread-like apperarance. In the youngest ring stages themitochondrion can be seen to conform to the circular shape of the parasite. Photographs immediately following. merozoite invasion were of insufficient quality for reproduction although the mitochondrion was readily visible to the eye. Part of the difficulty in obtaining high resolution photographs is that the parasites are alive after Rh123 staining and frequently move during photographic exposure. As the parasite matures through the trophozoite stage, the mitochondrion elongates and becomes proportionately wider (Fig. 1, b-e). In the transition from the trophozoite to schizont stage, the mitchondrion becomes distinctly branched (Fig. 1, f-k). This branching appears to radiate from spherically thickened areas along the organelle (Fig. 1, j). Throughout the period of nuclear division the mitochondrion branches extensively while increasing its overall dimensions. The process of branching produces dramatic variations in morphology and often the mitochondrion appears to be twisted upon itself. As cytokinesis begins, the mitochondrion becomes spoke—like with distal portions becoming incorporated into the newly formed merozoites (Fig. l, l—q). Each daughter merozoite receives a single mitochondrion, which extends almost its entire length (Fig. 1, r-s). Extraerythrocytic merozoites, 104 released from stained segmenting schizonts, also had distinctly fluorescent mitochondria. Over the entire cycle marked. parasite-to-parasite variation. existed. in the intensity of mitochondria-associated fluorescence. In no case was there fluorescence associated with the erythrocyte cytoplasm or with the food vacuole of the parasite. Rhodamine 123 accumulation and retention were examined in response to agents known to affect mitochondrial transmembrane potentials. In serum-free medium late trophozoite and schizont stages were exposed to selected inhibitors before of after staining. The effect of each inhibitor was determined at various time intervals on both mitochondrial and cytoplasmic fluorescence (Table I). In prestained parasites the protonophore, CCCP (10.6 M), and a -5 10 M concentration of the ATP synthetase inhibitor, DCCD, resulted in a rapid release of dye from the mitochondrion, followed by a slower release from the cytoplasm (Fig. 1, t). -6 Lower concentrations of DCCD (10 M) as well as incubation in the presence of the electron transport inhibitor antimycin Al (10-5 M) resulted in decreased mitochondrial dye retention without affecting cytoplasmic dye retention. -5 Pretreatment with 10 M CCCP had no effect on dye accumulation whereas pretreatment with either 10-6 M DCCD or antimycin Aj_ resulted 111 decreased Initochondrial fluorescence. When parasites were pretreated with a very low DCCD concentration (10"9 M), dye accumulation was increased selectively in the mitochondrion. This effect was TABLE I . 105 Effects of mitochondrial inhibitors on the accumulation or retention of Rh123. Effect on Effect on mitochondrial cytoplasmic Treatment Action fluorescence fluorescence CCCP H+ ionophore 10-6 M, 5 minb Diminished Increased 10-6 M, 20 min Diminished Diminished 10-5 M, 30 minC Unaffected Unaffected DCCD ATP synthetase inhibitor 10-5 M, 5 minb Diminished Unfected 10-5 M, 15 minb Diminished Diminished 10-6 M, 30 minb Diminished Unaffected 10-6 M, 30 min Diminished Unaffected 10-9 M, 24 hd Increased Unaffected Antimycin Al Electron transport inhibitor 10-6 M, 5 minb Diminished Increased 10.6 M, 30 minb Diminished Unaffected 10-6 M, 30 minC Diminished Unaffected a 1% hematocrit of 15-20% parasitemia of trophozoites and schizonts used in all experiments. b Prestained with 0.1 ug/ml Rh123 for 30 min, then treated with the inhibitor. C Pretreated with inhibitor, then after removal of compound stained as above. d Pretreated with inhibitor, then stained at 0.01 ug/ml Rh123, the lower Rh123 concentration was required to observe the effect. 106 apparent only when very low concentrations of Rh123 were used for staining. Treatment of prestained parasites with rotenone 6 -3 4 3 (5 x 10' M), NaN (10 M), HQNO (10' M), or KCN (10' M) 3 for 30 min resulted in the release of dye from the mitochondrion with no apparent effect on cytoplasmic dye 9 retention. Similar treatment with valinomycin (5 x 10- M), nigericin (10"7 M), DNP (10-4 4 -5 M), quercetin (10- M), or oligomycin (10 M) for 30 min resulted in the release of dye from both the mitochondrion and the cytoplasm, and only from the mitochondrion for lower concentrations of quercetin (10—5 M) and oligomycin (5 x 10_7M). DISCUSSION The mitochondrion is known to maintain a high transmembrane potential, and since Rh123 accumulation results from the maintenance of such a potential, this dye has been considered a specific marker for mitochondria (7, 15, 16). Accordingly, the morphological appearance, the pattern of development and segregation into merozoites, and responses to effectors of transmembrane potential convinc- ingly demonstrate the presence of a metabolically active mitochondrion throughout the erythrocytic cycle of P. falciparum. Although photographs represent only a two- dimensional View, while observing a wet mount preparation, all three dimensions of the motile parasite were revealed 107 - and in every case only a single mitochondrion was observable. The apparent lack of Rh123 accumulation by the ring forms may be due to quenching of the fluorescence by hemoglobin. These results also demonstrate the presence of a transmembrane potential across the parasite plasma and parasitophorous vacuolar membranes. A previous study using Rh123 with P. yoelii concluded that no mitochondrial function was present (32); however, in that study the investigator used a concentration of Rh123 that was IOO-fold greater than we used, resulting in high parasite fluorescence that possibly' obscured the presence of the mitochondrion. Previous studies on energy metabolism in mammalian malarial parasites indicate that glycolysis is the principle source of ATP (29). If the maintenance of a mitochondrial membrane potential is not for ATP, then the parasite is expending energy for another purpose. The mitochondrion may be involved in Ca++ regulation, a function commonly ascribed to this organelle (2). Calcium is knoun to accumulated in Plasmodium (33). Alternatively, as has been suggested, the mitochondrion may be associated with pyrimidine synthesis (10). It is also possible that this organelle is maintained throughout the erythrocytic cycle in order to provide mitochondria for the gametocyte stages, which possess cristate mitochondria (30). Our results using agents known to disrupt mitochondrial activity are similar to those observed in other eukaryotic 108 cells (7, 15) indicating that the intraerythrocytic stages of “P. falciparum 3possess. a mitochondrion having similar properties. Protonophores, such as CCCP, and electron transport inhibitors, such as antimycin A dissipate proton 1' electrochemical gradients-the former across any membrane and the latter across only the inner mitochondrial membrane (34). Prestaining the parasites with Rh123 followed by treatment with either of these agents resulted in an initial increase in cytoplasmic fluorescence, probably due to dye release from the mitochondrion. When incubation times were prolonged, cytoplasmic fluorescence returned to normal levels for antimycin A and was greatly reduced for CCCP. 1 The effects of valinomycin, which. were similar to CCCP treatment, indicate that the potential across the plasma membrane depends upon both a delta-psi and delta-pH component. The slower dissipation of the proton gradient associated with the cytoplasm may reflect its lower surface- to volume ratio compared to that of the mitochondrion. The same may be true for the time-dependent effect observed for DCCD (10'5)' Parasites pretreated with a low concentration of the ATPase inhibitor, DCCD (10"9 M), and subsequently stained with a low concentration of Rh123 exhibited increased mitochondrial dye accumulation. This response was as expected since ATPase inhibition prevents utilization of the proton gradient without affecting electron transport (34). The net effect is an increase in transmembrane potential, _i‘ 'w 109 resulting in greater dye accumulation. Differences in control and. DCCD-pretreated parasites were only' apparent when using a low concentration of Rh123; the finding that Rh123 also inhibits ATP synthetase (22) may account for this observation. Unexpectedly, mitochondrial and cytoplasmic dye retention were reduced when prestained parasites were treated with 10.5 M DCCD; at 10-6 DCCD, only mitochondrial dye retention was decreased. If we assume the effect of high DCCD concentrations to be specific, then to account for the differential responses observed for 10.5 M, 10"6 M, and 10"9 M DCCD, two separate mitochondrial ATPases must be present. One mitochondrial ATPase should function as as a synthetase and be most sensitive to DCCD while the other as a less sensitive hydrolase (11, 24); the ATPase(s) associated with the maintenance of the cytoplasmic transmembrane potential also differ in DCCD sensitivity from the mitochondrial enzymes. An equally plausible explanation is that the effect of high DCCD concentrations is nonspecific; DCCD is a reactive molecule and can nonspecifically interact with cellular components (21). The result of treatment with oligomycin (10.5 M) or quercetin was identical ix) DCCD 1J1 a dose-dependent fashion, supporting the hypothesis that a mitochondrial and cytoplasmic ATP hydrolase may exist. Since cytoplasmic fluorescence in the parasite was not 6 -6 affected, antimycin A (10- M) as well as DCCD (10 and 1 110 10-9 M) inhibit mitochondrial ATP sythesis, but do not alter energy-dependent potentials across the plasma membrane. Therefore, inhibition of mitochondria function must be incomplete, or if completely inhibited, the cytoplasmic membrane potential dissipates very slowly or else the mitochondrion. normally provides only part. of the energy required for cellular processes. In the latter case, metabolic pathways such as glycolysis must be sufficient to supply the energy required to maintain the potential across the parasite plasma membrane, which is negative inside, as previously demonstrated for .P. chaubaudi (23). It is important to remember that parasites generally have profound modifications of mitochondrial function (6). In certain parasitic helminths and protozoans, a portion of the TCA cycle is often reversed to produce succinate, which is subsequently utilized for substrate-linked phosphorylation (5,18). In Ascaris muscle mitochondria, malate dismutation has been demonstrated to yield ATP (26). The importance of the mitochondria in P. falciparum is exemplified further by the fact that all agents that affected Rh123 retention also inhibited parasite development, both protein and nucleic acid synthesis were immediately inhibited (Ginsburg et al., manuscript in preparation). Moreover, compounds that inhibit mitochondrial protein synthesis are also lethal to this parasite (6, 9). Although our findings leave little doubt that the mitochondrion of P. falciparum actively maintains a 111 high transmembrane potential, to accurately define its physiological role will require detailed biochemical studies using isolated organelles. Such studies should reveal parasite-specific processes which may be targeted as sites of action for new chemotherapeutic agents. ACKNOWLEDGEMENTS This work was supported by a grant from the UNDP/World Bank/WHOI Special Programme for Research. and Training in Tropical Diseases. This is Article No. 11252 from the Michigan Agricultural Experiment Station. 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