Entamoeba histolytica: I. Aerobic metabolism

EXPERIMENTAL

PARASITOLOGY

35,232-243

Entamoeba EUGENE Laboratory

of Parasitic National

(1974)

histolytica: C. WEINBACH

I. Aerobic AND

Metabolism1

LOUIS S. DIAMOND

Diseases, National Institute of Allergy and Infectious Institutes of Health, Bethesda, Maryland 20014 (Submitted

for publication

April

Diseases,

2, 1973)

WEINBACH, EUGENE C., AND DIAMOND, LOUIS ,S. 1974. Entamoeba histolytica: I. Aerobic Metabolism. Experimental Parasitology 35, 232-243. The respiration of intact trophozoites harvested from axenic cultures of Entamoeba histolytica was studied with the polarographic technique utilizing the Clark oxygen electrode. A typical QoZ value for the freshly harvested amebae was 1 patom oxygen/mg protein/hr. It was conclusively demonstrated that this parasite, a putative anaerobe, not only consumes oxygen when provided, but has a high affinity for the gas. Added glucose, galactose, and ethanol increased the respiratory rates, whereas other carbohydrates were without effect on the endogenous respiration. Intermediates of the tricarboxylic acid cycle, amino and fatty acids did not stimulate the respiration of E. histolytica. Inhibitors of the mammalian respiratory chain (cyanide, antimycin) as well as agents that inhibit enzymes catalyzing ,the tricarboxylic acid cycle (malonate, fluoropyruvate, fluoroacetate, fluorocitrate) had little effect on the endogenous or glucosesupported respiration. Alkylating agents (iodoacetamide, iodoacetate), cinnamate, and N-ethylymaleimide strongly inhibited the oxygen consumption of E. histolytica. The Emetine and Metronidazole, at concentrachemotherapeutic agents, Paromomycin, tions that inhibit growth in citro, did not restrict the respiration. Storage of the trophozoites at 4 C led to progressive deterioraion of the parasites and loss of endogenous and glucose-supported respiration. The deterioration was paralled by loss of SH-materials from the amebae. Likewise, sonication or lysis with detergents abolished both the endogenous respiration and response to glucose. Exogenous NADH or NADPH evoked only marginal increases in oxygen consumption of the freshly harvested amebae, but were effective respiratory substrates with stored or sonicated organisms. Addition of vitamin Ks greatly enhanced the cndogenous and glucose-supported respiration of the intact amebae, as well as enhancing the response of stored or sonicated amebae to reduced pyridine nucleotides.

INDEX DESCRIPTORS: Metabolic inhibitors; Respiration.

Entamuoeba histolytica; Axenized amebae; Metabolism aerobic; Glucose; Galactose; Ethanol; NADH, NADPH; Vitamin K,;

The metabolism of E&amoeba histolytica has been presumed to be primarily anaerobit both in viva and in vitro. The basis for this presumtion is that the normal environ-

ment of the parasite is essentially anaerobic, and that a low redox potential is required for its optimal growth in culture (Neal 1967). N evertheless, various reports have indicated that E. histolytica may have an aerobic metabolism (for review, see Danforth 1967). These earlier studies were handicapped by the lack of axenic cultures

1 Presented in part at the Nineteenth Annual Meeting of the American Society of Tropical Meditine and Hygiene, San Francisco, California, November 14, 1970. 232 Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.

AEROBIC METABOLISM OF Entamoeba

which made the interpretation of the results uncertain. The availability of axenic amebae cultures (Diamond 1968) now provides the opportunity of conducting definitive biochemical studies on amebae in the absence of bacterial and other protozoan associates. Wittner ( 1968), studied the growth characteristics of axenic strains of E. histolytica and observed an oxygen gradient in culture medium containing the axenized amebae. From this indirect evidence he suggested that the parasite utilized oxygen. Wittner’s suggestion was of particular significance to us in view of our long term interests both in the mechanisms of aerobic energy metabolism (for references, see Weinbach and Garbus 1969) and parasite respiration (cf. Weinbach and Eckert 1969; Weinbach and von Brand 1970). The present report provides conclusive evidence that E. histolytica consumes oxygen when provided, and delineates some of the characteristics of the aerobic metabolism of this parasite. Future reports will summarize ‘other aspects of the biochemistry of E. histolytica. MATERIALS AND METHODS

Parasites. Amebal trophozoites of the HK-9 strain of E. histolytica were cultivated under axenic conditions in ,a stationary culture system consisting of 16 x 125 mm, screw-capped tubes containing 15 ml TP-S1-monophasic medium ( Diamond 1968 ) . After an incubation period of 72 hr at 35.5 C, the cultures were harvested and prepared for assay as follows: Each tube was chilled 5 min in an ice-water bath, inverted several times to loosen the amebae adherent to the walls of the tube, and centrifuged for 5 min at 850 g, in an International Centrifuge (Model UV). The supernatant fluid was decanted and the sedimented amebae suspended in 1 ml of chilled buffered saline, (0.11 M NaCl, 0.016 M K2HP04, and 0.003 M KHzPOd, pH 7.45), pooled in 35 ml graduated, conicaltipped, screw-capped centrifuge tubes and recentrifuged for 5 min at 850g. The super-

histolytica

233

natant fluid was ,aspirated and discarded; the amebae were suspended in fresh, chilled buffered saline (1 vol amebae/l4 vol buffer) and again centrifuged for 5 min at 850g. After this second washing and removal of the supernatant fluid, the amebae were suspended in fresh saline media so that each milliliter contained approximately 5.7 (4.5 7.5) x lo6 organisms/ml as determined with a Coulter Counter ( Model B ) ; which was equivalent to approximately 5.0 (4.3-6.3) mg protein/ml. Procedures. Oxygen uptake was determined polarographically with the Clark oxygen electrode (Yellow Springs Instrument Co., Inc., Yellow Springs, OH). One milliliter of the amebal suspension, diluted to the desired concentration (see legends to individual experiments), was used for each polarographic assay. The suspensions were incubated at 35 C in a glass cuvette that was mounted in a plastic, thermostated chamber and fitted with a magnetic stirrer. After incubation of the suspension at 35 C for 2 min to equilibrate with air, the electrode was inserted forming an air tight seal, and recording begun. Additions to the suspension in volumes not exceeding 50 ~1 were made with microliter syringes through a capillary port in the cuvette. The instrument was calibrated with NADH and rat liver mitochondria as described by Chappell (1964) and Estabrook (1967). Protein was determined by a modification of the biuret procedure (Szarkowska and Klingenberg 1963) after treatment of the amebal suspension with 0.5% sodium deoxycholate. Crystalline bovine serum albumin was used ,as standard. Sulfhydryl compounds were estimated by the method described by Ellman, et al. ( 1961), with cysteine as the standard. Glass redistilled water was used to prepare solutions of all reagents, which were of the highest purity available commercially. RESULTS

Respiration of E. histolytica. Figure 1 depicts the results of a typical polaro-

234

WEINBACH

AND

DIAMOND

PARASITES

FIG. 1. Oxygen uptake of Entamoeba histolytica trophozoites, Mesocestoides corti larvae, and rat liver mitochondria. (A) The cuvette contained 2.5 X lo6 organisms/ml. The arrow indicates addition of 10 pmoles of D-glucose. The numbers on the tracing express the oxygen consumption in nanoatoms per minute. (B ) lower curve. The cuvette contained 0.2 ml of M. corti l’arvae suspended in 0.8 ml of Tyrodes solution ( Weinbach and Eckert 1969). (B ) upper curve. The cuvette contained 1.0 ml of a water-lysed preparation of rat liver mitochondria (0.4 mg protein) isolated and assayed as described by Schnaitman et al. ( 1967). The arrow indicates addition of 45 nmoles of cytochrome c ( CYTO c), Assay temperatures were: E. histolytica (35 C), M. corti (37 C), and rat liver mitochondria (24 C).

graphic experiment. Figure 1A shows that freshly harvested trophozites of E. histoZytica consume oxygen at a substantial rate. The endogenous rate in this experiment was 42 nanoatoms of oxygen per 2.5 million organisms per minute, or an equivalent QoZ of approximately 1 patom oxygen/mg protein/hr. Addition of 10 m&l glucose increase the oxygen consumption three fold. Although the addition of glucose to freshly harvested, intact trophozoites always stimulated respiration, the magnitude of the stimulation varied from preparation to preparation. The rate of oxygen consumption was proportional to the number of amebae present in the reaction chamber. Not ‘only do these parasites consume oxygen when provided, but the organisms have a high affinity for this gas. This is shown by the shape of the polarographic trace. As oxygen is consumed in the sealed cuvette, its concentration diminishes progressively until the ‘oxygen level approaches zero. As indicated by the arrow in Fig. lA, the rate of oxygen consumption by E. histolytica remained unchanged even at extremely low oxygen tensions. In contrast, the respiratory rate of many other parasites is dependent upon the oxy-

gen tension (reviewed by von Brand 1966). For example, as shown in Fig. lB, the rate of oxygen consumption by the larvae of Mesocestoides corti declined as the oxygen tension decreased (cf. Weinbach and Eckert 1969 ) . It may also be seen in Fig. 1B that the rate of oxygen consumption by isolated rat liver mitochondria-considered the hallmark of aerobic metabolism-exhibits similar kinetics to that shown by the active trophozoites of E. histolytica. It must be emphasized that it is the shape of the polarographic tracings that should be compared, and not the absolute rates of respiration. %.&&ate utikzation. D-Glucose was a prime exogenous substrate for supporting oxygen consumption by E. histolytica (Table I). Of the other carbohydrates tested.. only galactose unequivocally enhanced the respiration. Activities shown for the other carbohydrates were either marginal or could be attributed to glucose contamination of the commercia1 substrate used.* 2 All carbohydrates used in this study were tested enzymatically (Slein 1963) for possible contamination with D-glucose.

AEROBIC

METABOLISM

OF

All substrates were tested as illustrated in Fig. 2 with n-fructose. The addition of this hexose had little effect on the respiration of the parasites. The subsequent addition of n-glucose, which stimulated respiration more than two-fold, served as a control. This enabled us to ascertain whether the organisms were still viable, or whether the substrate being tested was inhibitory. The addition of various amino and fatty acids were without effect on the endogenous respiration. The only exception was that L-serine markedly stimulated the oxygen consumption. This unexpected observation is being investigated further and will be the subject of a future communication. Intermediates of the tricarboxylic acid cycle, such as pyxuvate, isocitrate, suc&ate, malate, and a-ketoglutarate, added alone or in combination, did not stimulate respiration. The addition of detergents such as deoxycholate or Lubrol WX 3 to increase 3 ICI America,

Inc., S’tamford,

Entamoeba

histolytica TABLE

Iiespiration

235 I

of Entamoeba histolytica

Substrate

Carbohydrat,es D-Glucose D-Galactose D-Fructose D-ltibose : Maltose D-Mannose D-Xylose Glycerol nr,-Glycerol-3-phosphate 2-Deoxy-n-glucose

Oxygen uptake Relative maximal activity”

100 6X 30 30 27 10 0 0 0 0

Amino acids

0

Fatty

0

acids

Tricarboxylic acid cycle intermediates Miscellaneous

: Acetaldehyde Choline Ethanol

0 0 0 61

Connecticut.

permeability of the protozoan membrane did not evoke respiration with these substrates. Addition of 1 mM NAD also had no effect in these experiments. It may be seen in Table I that addition of 10 mM ethanol enhanced the oxygen consumption of E. histolytica. Increases in ethanol concentration ( 173 mM) further stimulated both the endogenous and the glucose-enhanced respiration. Ethanol at ex-

u Based on the rates found with n-glucose as 100%. The average number of organisms used in these assays was j.0 X 106: equivalent to 4.5 mg protein/ ml. All subst,rates were added in a concentration of 10 mM of the active form. The amino acids tested were : alanine, arginine, cysteine, glycine, glutamic, glutamine, glutathione, histidine, lysine, leucine, proline, methionine, sarcosine, threonine, tyrosine and valine, Both the DL- and L-forms of the acids were used. The fatty acids tested were: acetic, butyric, @-ntihydroxybutyric, caproic, lactic, octanoic, and proprionic.

PARtSITES MEDIUM

FRUCTOSE

20 mM

FIG. 2. Oxygen uptake of Entamoeba histolytica trophozoites in presence of carbohydrates. The cuvette contained 2.0 x 10’ organisms/ml. The arrows indicate additions of 20 mM D-fructose and 20 m&f D-glucose, respectively. The numbers below the tracing express the oxygen consumption in nanoatoms per minute.

236

WEINBACH

AND

tremely high concentrations, however, (50 ~1 of “Absolute”; 865 m M ) was inhibitory. It is of interest that ethanol is ,an endprodTABLE

II

Effect of Inhibitors on the Respiration Entamoeba histolytica Inhibitor

Final concn WO

Inhibition ye initial rate” Endogenous

Cyanide Rotenone Amytal Oligomycin AnGmycin 2,4-Dinitrophenol Thenoyltrifluoroacetone p-Chloromercuribenzoate N-Ethylmaleimidec Iodoacetamidec 2-Deoxy-n-glucose Cinnamate Dicoumarol Malonate Fluoroacetate Fluorocitrate Fluoropyruvate Arsenate Arsenite Paromomycin Emetine Metronidazole Deoxycholate Boiledd Sonication”

5.0 0.02 2.0 30 b&n1 30 bdml 0.01 3.0 0.5 1.0 50.0 30.0 20.0 0.5 25.0 50.0 2.5 Fi.0 2.5 5.0 2.5 5.0 5.0 5.0 10.0 2.0 4.0 4.0 O.l2c/;,

oj

D-Ghcase*

0 0 0 0 0 0

0 0 0 0 0 0

0

0

0 57 73 0 100 34 0 21 0 0 0 0 0 0 9 12 0 20 35 f 100 100 100

0 63 72 0 100 46 0 27 0 0 7 14 0 23 12 18 0 20 32 f 100 100 100

The average number of organisms used per assay was 6 X 106: equivalent to an average protein content of 5.6 mg/ml. a Average values of at least two independent determinations. b Added in a final concentration of 10 m&f. c Inhibition observed 3 min after addition of inhibitor. d 10 min at 100 C. e 10 set at 3.6 amp, Power Setting No. 4, microprobe, Branson Sonifer, Model S75.

DIAMOND

uct of the aerobic metabolism of this parasite ( Montalvo, et al. 1971). It should be emphasized that all of these experiments were done with freshly harvested trophozoites. As will be shown below, storage of the parasites has a profound influence on their respiration. Effect of inhibitors. These results are summarized in Table II. Each inhibitor was tested independently on the rate of endogenous and glucose-supported respiration, and essentially similar results were obtained under either condition. Cyanide, in concentrations as high as 5 mM had no effect on the respiration ‘of E. histolytica. Other respiratory inhibitors such as antimycin, rotenone, and oligomycin 4 also were ineffective. 2,4-Dinitrophenol and arsenate, which block respiratory-chain and substrate-linked phosphorylations, respectively ( Slater 1963 ), had no effect in these experiments. Inhibitors of the enzymes catalyzing the tricarboxyhc acid cycle, malonate, fluoroacetate, fluorocitrate, fluoropyruvate, and 4 It was necessary to dissolve these compounds in ethanol and appropriate controls were done with ethanol alone.

PARA+SITES MEDIUM N-ETHYLMALEIMIDE

1mM

I-1 mid

02=0

FIG. 3. Effect of N-ethylmaleimide on the respiration of Entamoeba histolytica. The cuvettes contained 5.5 X 10’ organisms (4.9 mg protein) per assay. The arrow indicates addition of 1 mM N-ethylmaleimide. After completion of the inhibitor experiment, the chart paper was returned to the starting point and the control experiment done. The numbers below the tracings express the oxygen consumption in nanoatoms per minute.

AEROBIC

METABOLISM

OF

Entamoeba

histolytica

237

PAR+nSlTES MEDIUM Arrows indicate 25 mM additions of each Inhibitor

IODOACETAMIDE

! I

I 2

I 3

I 1 4 5 MINUTES

I 6

I 7

I 8

I 9

FIG. 4. Effect of iodoacetamide and iod’oacetate on the oxygen consumption of Entamoeba histolytica trophozoites. The cuvette cntained 5.8 X 10’ organisms/ml in each assay. The numbers on the tracings express the oxygen consumption in nanoatoms per minute.

arsenite (cf. Gordon, et al. 1967) were only partially inhibitory at high concentrations. Thenoyltrifluoroacetone, a potent inhibitor of mammalian succinate oxidase (Tappel 1960) and cestode glycerol-Sphosphate oxidase (Weinbach and von Brand 1970), did not restrict the respiration of the amebae. It is likely that the inactivity of some of these reagents is owing to permeability barriers of the intact cells (see Webb 1963). This point is illustrated with the sulfhydryl reactants. The inactivity observed with pchloromercuribenzoate (presumably can be ascribed to its inability to penetrate the cells and reach the active sites of the oxidative enzymes. Likewise, the addition of Nethylmaleimide (Webb 1966) to the respiring trophozites had no immediate effect, but progressive inhibition was observed with time (Fig. 3). The alkylating agents iodoacetamide and iodoacetate at high concentrations were effective inhibitors. It may be seen in Fig. 4 that iodoacetamide was more rapid in its action than the corresponding acetate ion, presumably reflecting difference in cellular penetration of the two forms of this sulfhydryl reactant. Cinnamate, an inhibitor of glycerol-3phosphate dehydrogenase in rat liver homogenates (Sacktor ,and Dick 1965), and

in Crithidia fasciculata (Bacchi et al. 1968), and of glycerol-3-phosphate oxidase in cestode mitochondria (Weinbach and von Brand 1970), also inhibited the respiration of E. histolytica. The addition of the undissociated compound in ethanol resulted in a drop in pH (5.2) of the amebal suspension. Although the dissociated cinnamate ion is not an effective inhibitor at pH 7.4, the complete inhibition observed at pH 5.2 is not a result of the change in pH alone. Lowering the pH of the suspension to 5.2 by other means had much less influence on the respiration. The effects of three chemotherapeutic agents on the respiration of the trophozoites are summarized in Table II. Paromomycin had no effect, Emetine was mildly inhibitory, and Metronidazole usually had no effect ‘on the oxygen consumption of the parasites. Because IMetronidazole slightly stimulated ( 1.5~ ) the respiration of some preparations of E. histolytica, it was tested in these concentrations with isolated rat liver mitochondria. It did not release respiratory control or uncouple oxidative phosphorylation in mitochondria. Effect of storage. All ‘of the experiments described ,above were done with freshly harvested, motile trophozoites. Storage of the parasites at 4 C during the course of the polarographic determinations resulted

238

WEINBACH

in progressive loss of the endogenous respiration, and a diminished response to exogenous glucose. The deterioration was

x300.

Freshly

harvested

DIAMOND

even more marked if the washed trophozoites were stored at 35 C. These changes usually occurred during a 2-3 hr period.

changes in trophozoites of Entamoeba histolytica during stortrophozoites. (6) After 2.5 hr storage at 4 C. Phase microscopy.

FIGS. 5 and 6. Morphological

age. (5)

AND

AEROBIC

METABOLISM

OF

Examination of the trophozoite suspension by phase contrast microscopy revealed that gross changes in morphology and deterioration of the parasites (lysis) had occurred during storage (Figs. 5 and 6). Sonication or addition ,of detergents to the fresh suspensions also abolished both endogenous respiration and response to glucose (Table II). Pyridine nuc1eotid.e oxidation and vitamin KS. Freshly harvested trophozoites were incapable of oxidizing exogenous NADH or NADPH to any appreciable extent, a finding analogous to observations with intact mammalian mitochondria (for review, see Wainio 1970). The addition of vitamin K3 resulted in a rapid uptake of oxygen (Fig, 7). Additional tests disclosed that this increased rate was owing Iargely to vitamin I& alone and not to oxidation of the added nucleotide. Addition of vitamin I& to stored parasites, however, resulted in a feeble rate of oxygen uptake which was greatly enhanced by subsequent addition of NADPH (Fig. 8). Addition of a second portion of NADPH indicated that the decline in oxygen consumption was not related to deplePARASITES MEDh4

‘;“:,.. -f

min+

FRESHLY

FIG. 7. Effect of NADPH

ISOLATED

and vitamin K, on the uptake of freshly harvested trophozoites of Entamoeba histolytica. The cuvette contained 3.8 x 108 organisms. Arrows indicate additions of 1.0 pM NADPH and 0.5 pA4 vitamin IL The numbers below the tracing express the oxygen consumption in nanoatoms per minute. oxygen

histolytica

PARA+SITES

VITAMIN

MEDIUM

1

pl

mind

239

K3

3 HRS AFTER

NADPH

ISOLATION

FIG. 8. Effect of vitamin & and NADPH on the oxygen uptake of stored trophozoites of Entamoeba histo&ica. The cuvette contained 7.6 X 108 drganisms which had been stored at 4 C for 3 hr after harvesting. Arrows indicate additions of 0.5 pM vitamin IG and 1.0 pM NADPH each. The numbers below the tracing express the oxygen consumption in nanoatoms per minute.

tion or destruction of the nucleotide. Analogous results were obtained with NADH as substrate. It may be seen in Fig. 9 that NADH oxidation in the stored parasites stimulated by vitamin K, was only partially sensitive to rotenone. Likewise, NADH or NADPH oxidation was weakly inhibited by dicoumarol or by thenoyltrifluoroacetone.5 Sulfhydryl content. During the course of this study we observed that the sulfhydryl content of the trophozoites decreased during storage (Table III). With freshly harvested parasites, a major portion of the total amount of SH material in the trophozoite suspension was recovered in the pellet after centrifugation. After 3 hr of storage the situation was reversed; now the major portion of SH material was found in the supernatant fluid. Whether the SH material reacting with Ellman’s reagent represents an intrinsic constituent, or substances engulfed by the parasite during growth (cf. Schneider

\/ITAMINK3

IdO natoms oxygen

&I

Entamoeba

5 A preliminary account of the NADH and NADPH diaphorase activities in E. hitioh~tica was presented at the Forty-seventh Annual Meeting of The American Society of Parasitologists, Miami Beach, Florida, November 6-10, 1972 (Weinbach and Diamond).

240

WEINBACH

AND

DIAMOND

PARASITES

ROTENONE

IO pM

f 100“atomsoxygen I

FIG. 9. Effect of NADH and rotenone on the Entumoeba histolytica. The cuvette contained 3.8 X at 4 C for 6 hr. Arrows indicate additions of 1.0 pM rotenone each. The numbers below the tracing atoms per minute.

and Gordon 1968) is not known. However, the correlation with the decrease in respiratory activity with time (Table III) suggests that this material may be an endogenous TABLE Change in Sulfh,plryl Time of storage (hr)

oxygen uptake of stored trophozoites of 10” organisms/ml which had been stored pM NADH; 0.5 pM vitamin Kg and 10 express the oxygen consumption in nano-

constituent of the parasite. Its release into the medium parallels the deterioration of the parasite as revealed by the morphological changes described above. Likewise, III

Content of Entamoeba histolytica SH content

Fraction

nmoles/ml

During

Storage

Respiration natjoms/oxygen/min/ml %‘” Endogenous

0

1

2

3

Totalb Supernatant Pellet

289 33 247

12 88

Total Supernatant Pellet

281 86 173

33 67

Total Supernatant Pellet

248 124 133

4x 52

Total Supernatant Pellet

“4Q 145 102

59 42

Glucose

74

1.50

67

131

37

48

26

x2

At the hours indicated, l.O-ml samples of the amebal suspensions were sonicated (Branson Sonifer, Model No. S75, Power Setting No. 4 for 30 set, microprobe), and portions were analyzed for SH-reacting material l.O-ml samples of the original suspensions were centrifuged at 755 g for (Ellman, et al. 1961). Additional 10 min, and the supernatant fractions were analyzed. The pellets were resuspended in 1.0 ml of the buffered saline medium, sonicated as above, and portions were analyzed for SH content. The data are average values of three independent experiments, and the original suspensions contained approximately 6.0 X lo6 organisms (6.5 mg protein) per ml. QCalculated from the total amount of SH material recovered. b Total amount of SH material found.

AEROBIC

METABOLISM

OF

the marked decline in the vitamin Ka-stimulated respiration shown above (Fig. 8) appears to be related to the loss of SH material from the parasite. Cysteine reacts nonenzymatically with vitamin KS, but the rate of oxidation of the reduced vitamin falls off rapidly. Addition of cysteine to the trophozoites did not improve their stability during storage. Cataluse activity. Attempts to demonstrate catalase activity by the polarographic method previously described ( Weinbach and von Brand 1970) were negative. These experiments were done with intact, sonicated, and detergent-lysed amebae. DISCXJSSION

The results of this study provide cogent evidence that freshly harvested, axenically, grown trophozoites of E. histolytica not only consume oxygen when provided, but have a high affinity for this gas. The most likely endogenous substrate is carbohydrate. The parasite has ample endogenous glycogen (Rosenbaum and Wittner 1970; FeriaVelasco and Trevino 1972), and of the various classes of exogenous substrates tested, carbohydrates (especially glucose and galactose) markedly stimulated respiration (Table I). Likewise, inhibitors of glycolysis were effective in restricting the endogenous respiration ( Table II ). Studies with respiratory inhibitors (for review, see Biicher and Sies 1969) indicated that enzymes of the tricarboxylic acid cycle are not operative in E. histolytica (Table II). Nor is there any evidence that a functional respiratory chain, analogous to that found in mitochondria of eukaryotic cells, is present in the trophozoites of E. histolytica. Although NADH and NADPH when added alone to the stored parasites stimulated oxygen consumption, the effect was minimal in the absence of added vitamin KS. Furthermore, relative insensitivity to rotenone and to dicumarol suggests that the reduced nucleotides are oxidized by flavoproteins, perhaps similar to the “DT

Entamoeba

histolytica

241

diaphorase” described by Ernster et al. (X362), rather than via the respiratory chain. These results are wholly compatible with the ‘absence of mitochondria in E. histolytica (Rosenbaum and Wittner 1970). From the point of view of comparative biochemistry, it is of interest that many of these observations with inhibitors are similar to the effects observed by Hendler et al. ( 1969) in their studies of the respiratory chain of Escherichia coli. This does not imply, however, that the respiratory chains of the two organisms are similar. The lack of any pronounced effect by the chemotherapeutic agents, Paromomycin, Emetine, and Metronidazole, in concentrations that inhibit growth of axenized E. histolyticu in vitro (Diamond and Bartgis 1971) shows that interference with respiratory metabolism is not the basis of their pharmacological action. Neither does the enhanced respiration evoked by Metronidazole appear to be related to uncoupling of oxidative phosphorylation or release of respiratory control (Weinbach and Garbus 1966). The cause of the inconsistent effects observed with Metronidazole on the respiration of E. histolytica is not known, nor has it been studied in detail. Identification of the specific enzymatic sequence responsible for the observed oxygen consumption of E. histolytica has not been ‘attempted in this study. Montalvo et al. ( 1971) in a recent investigation of monoxenic cultures of E. histolytica found that the principal products of aerobic metabolism were COa, acetate, and ethanol. If the same pattern of metabolism occurs in axenically grown E. histolytica, then oxidative decarboxylation of pyruvate (arising from glucose via glycolysis) could account for a substantial portion of the observed oxygen consumption and product formation. We observed the formation of ethanol and the absence of lactate as an endproduct of aerobic metabolism in the axenic cultures. The biological significance of respiration

242

WEINBACH

AND DIAMOND

to E. histolytica, whose normal habitat is putatively anaerobic, remains to be elucidated. From the results and considerations presented above, it appears unlikely that oxygen is used directly for energetic purposes. Undoubtedly, E. histolytica, in common with many other parasites, meets its energy demands largely by glycolytic processes (see review by von Brand 1966). A portion of the reducing equivalents, however, such as NADH formed during glycolysis, may be oxidized directly by a flavoby transhyprotein oxidase, or indirectly drogenase activity, in ‘order that glycolysis may proceed. Alternatively, NADH, formed during glycolysis, also may be reoxidized by reducing acetaldehyde to ethanol, one of the endproducts of aerobic metabolism in E. histolytica (cf. Montalvo et al. 1971). Respiration may be utilized as a detoxification mechanism. It is possible that oxygen is toxic for the parasite in viva, and that the oxidative enzymes serve as scavengers to remove oxygen from the immediate environment. Although the high affinity of the trophozoites for oxygen at low tensions is compatible with this suggestion, there is no direct evidence on this point. Finally, it should be considered that a portion of the oxygen consumption may be utilized for biosynthetic purposes. The oxygen requiring synthesis of collagen in adult Ascaris Zumbricoides (Fairbairn 1970) illustrates this possibility. Regardless of these considerations, our study demonstrates unequivocally that axenically-cultivated E. histolytica has the capacity for utilizing oxygen in vitro. The finding that the ‘parasite has a high affinity for oxygen at low tensions is of particular significance in view of the normal habitat of the amebae, and indicates that aerobic, as well as anaerobic, processes may be operative in vivo. ACKNOWLEDGMENTS The authors express their deep appreciation for the superb technical help of h4r. C. Elwood Claggett and Mrs. I. Louise Bartgis. We are indebted

to Dr. James A. Dvorak of Figs. 56.

for the photomicrographs

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