Molecular and Biochemical Parasitology, 17 (1985) 1-18 Elsevier
1
MBP 00585
MINI REVIEW THE STRATEGIES OF ENERGY CONSERVATION IN H E L M I N T H S
PETER K(~HLER Department of Parasitology, University of Zfirich, Winterthurerstr. 266a, 8057 Ziirich, Switzerland (Received 18 April 1985)
Key words: Energy conservation; Substrate level phosphorylation; Electron transport phosphorylation; Respiration; Fermentation; Ascaris suum; Fasciola hepatica
INTRODUCTION
Research on the bioenergetics in parasitic worms has become a fascinating field in comparative biochemistry and it has been repeatedly speculated that the differences observed between energy conserving pathways of helminths and their vertebrate hosts may be exploited for the design of chemotherapeutic agents directed against worm infections [1,2]. The general principle of biological energy conservation consists primarily of a system of oxidation-reduction reactions which are coupled to an ATP synthesizing enzymatic step. In substrate level phosphorylation (SLP) part of the free energy contained in a degradable substrate is initially conserved in an 'energy-rich' intermediate from which it is transferred to the universal energy carrier ATP (Fig. 1, left). The alternative principle of energy conservation proceeds through electron transport-associated phosphorylation (ETP) in which an electrochemical gradient of protons serves as an intermediate thermodynamic force that drives ADP phosphorylation to form ATP (Fig. 1, right). While all reactions involved in SLP are catalyzed by soluble enzymes, the complex enzymatic systems responsible for ETP are membrane-bound and the precise nature of this phosphorylating process is still not well understood. THE GENERAL PATHWAYS OF ENERGY CONSERVATION IN HELMINTHS
As in all living cells energy conservation in helminths is achieved through the basic Abbreviations: SLP, substrate level phosphorylation; ETP, electron transport-associated phosphorylation; PEP, phosphoenolpyruvate. 0166-6851/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)
Electron donor /
X-P
Kinase
ADP
• X
2el
~ ~ f i H *
"
l
ATP
~
ADP
,, P,
ATP
÷
H20
Electron acceptor
Fig. I. Schematic representation of the principles of (left) SLP and (right) ETP. X~P phosphate bond: A~.u+ -- trans-membrane elcclrochemical proton gradient,
"energy-rich'
concepts o f either SLP or ETP. In comparison to higher animals, helminths are, however, usually equipped with a higher n u m b e r o f different metabolic strategies to accomplish A T P formation via the two phosphorylating principles. As summarized in Table I, helminths possess a variety of anaerobic fermentations in which A T P synthesis occurs exclusively by SLP. Other energy conserving pathways functioning in the absence of oxygen are sometimes referred to as anaerobic respirations. These reactions play an i m p o r t a n t role in helminth bioenergetics and are coupled to electron transport-dependent synthesis of ATP. In addition, helminths contain aerobic respiratory chains in which A T P is conserved by E T P and in some helminths an oxygen-dependent respiration may drive a SLP-associated redox reaction. While the latter type of aerobic fermentation is unequivocally present in some p r o t o z o a n parasites [3,4] their occurrence in helminths remains to be determined. Representative examples for the various types of fermentations and respirations will be subsequently discussed. As substrates for energy conservation helminths use primarily c a r b o h y d r a t e from which glycogen is the major storage polysaccharide, consisting in m a n y cases of as much as 10% of the w o r m ' s wet weight [5]. At least in adult helminths the utilization o l fatty acids as an energy source is precluded and the degradation of carbohydrates and a m i n o acids b e y o n d the acetate step is only barely feasible. This metabolic insufficiency has been explained by the low activity or lack of enzymes involved in the pathways of terminal oxidation, in particular those of the tricarboxylic acid cycle, 13-oxidation pathway and the c y t o c h r o m e c oxidase-linked respiratory chain. As an alternative, helminths predominantly use fermentations and anaerobic electron transport reactions to generate chemical energy in the form of ATP. M a r k e d oxidative characteristics
TABLE 1 Metabolic strategies for ATP conservation in helminths Pathway
Principle of phosphorylation
ATP/2e
Anaerobic fermentation Anaerobic respiration Aerobic fermentation Aerobic respiration
SLP ETP SLP ETP
I 1 1
1-3
appear to be restricted to the outermost tissue layers of adult worm species, to some small helminths and to larval and other intermediate stages [6,7]. It is interesting to note that carbohydrate degradation in helminths inhabiting the blood vessels and subcutaneous tissues was found to be primarily mediated by the simple linear metabolic strategy of glycolysis with lactate as predominant or sole end product [8]. In the majority of worms catabolism is, however, branched and thus of a variable thermodynamic efficiency. Such types of metabolic pattern occur in a great variety of intestinal worms and in flukes inhabiting the bile ducts of vertebrate livers [5,8]. Characteristically, in these parasites ,carbohydrate is often converted to a mixture of volatile acids, succinate, lactate and carbon dioxide. Some species metabolize carbohydrate to form alcohols or other more rarely occurring organic end products. Unfortunately, detailed and continuous studies on the bioenergetics of helminths are essentially limited to two of the most easily obtainable species, the nematode Ascaris suum and the liver fluke Fasciola hepatica. I, therefore, would like to restrict the discussion primarily to the studies obtained with these two model systems. Interestingly, these investigations have uncovered a type of bioenergetic strategy shown to be widely distributed among helminths, but they do not allow a complete generalization to be made, as it is apparent that metabolism does vary from species to species and in some instances differs remarkably from that observed in the two most extensively studied species. ENERGY CONSERVATION ASSOCIATED WITH FERMENTATIONSAND ANAEROBIC ELECTRON TRANSPORT PATHWAYS It is now generally accepted that the bioenergetic pathways found in the adult stages of ascarid worms, liver flukes and many other helminths function predominantly anaerobically. Interestingly, a further characteristic feature of these worms is that the major pathway of carbohydrate degradation involves carbon dioxide fixation into glycolytically formed phosphoenolpyruvate (PEP) by PEP carboxykinase to give oxalacetate (Fig. 2) [6,8]. Unlike the host tissues, the parasite is not capable of forming significant quantities of cytoplasmic pyruvate, since the enzyme pyruvate kinase is present in very low or even barely detectable amounts. Other factors apparently determining the carbon flow in the direction of dicarboxylate formation are (1) a high affinity of the carboxykinase for its substrate PEP; (2) the high concentrations of PEP and carbon dioxide prevailing in the parasite tissues; and (3) the presence of an extremely active cytoplasmic malate dehydrogenase which serves to reduce the oxalacetate formed by carbon dioxide fixation. As demonstrated in Fig. 2, cytoplasmic malate formation is thus basically comparable to lactate fermentation. During the last two steps of both pathways 'energy-rich' phosphate is regenerated by kinase enzymes and dehydrogenases serve to reoxidize the pyridine nucleotide reduced during the middle phase of glycolysis.
2
Glucose
NAD1÷ NADH ~=""
1
2 PEP ME
Ma k~,te PYR
Pyr Hvate - -
OAA
'~bl]
i' [ H I
1[ ' FUM
FH~!/J r Llle
--
SDH))FR Lactate
Malate
~ J (]CHla[e
_J
I
1 NADH-
DH
1 " RO
O:. m202
Fig. 2. Glycolysis and CO~-tixation in helminths. PEP = phosphoenolpyruvate: PYR = pyruvate: OAA = oxalacetate. Fig. 3. Anaerobic malate dismutation in helminth mitochondria. FUM = fumarate hydratase; ME -- malate dehydrogenase (decarboxylating); PDC = pyruvate dehydrogenase complex; SDH = succinate dehydrogenase; FR = fumarate reductase: NADH-DH = NADH dehydrogenase; RQ = rhodoquinone (after K6hler and Bachmann [12]). In Ascaris, liver flukes a n d o t h e r helminths, m a l a t e derived from c a r b o n dioxide fixation b e c o m e s the key m i t o c h o n d r i a l s u b s t r a t e [6,9]. A f t e r p e n e t r a t i n g the mitoc h o n d r i a this c o m p o u n d u n d e r g o e s an o x i d a t i o n - r e d u c t i o n r e a c t i o n (Fig. 3). One part is o x i d a t i v e l y d e c a r b o x y l a t e d to yield p y r u v a t e , r e d u c e d p y r i d i n e n u c l e o t i d e a n d c a r b o n dioxide. In Ascaris muscle the malic enzyme ( m a l a t e d e h y d r o g e n a s e d e c a r b o x y l a t i n g , EC I. I. 1.39) c a t a l y z i n g this reaction is unusual in that it reacts preferentially with N A D + r a t h e r t h a n N A D P + [ 10]. As also s h o w n in Fig. 3, the r e d u c i n g p o w e r g e n e r a t e d in the o x i d a t i v e b r a n c h serves then to reduce a n o t h e r p o r t i o n of m a l a t e via f u m a r a t e to succinate. This p a t h w a y was f o u n d to p r o c e e d t h r o u g h a m i t o c h o n d r i a l m e m b r a n e - b o u n d electron t r a n s p o r t system involving a N A D H - d e h y d r o g e n a s e , a q u i n o n e a n d a t e r m i n a l e n z y m e r e d u c i n g f u m a r a t e to succinate [ 11]. R e g a r d i n g the final f u m a r a t e reducing r e a c t i o n in h e l m i n t h m i t o c h o n d r i a , it is still not c o m p l e t e l y clear w h e t h e r this s h o u l d be a s c r i b e d to a s e c o n d f u n c t i o n a l aspect o f a m a m m a l i a n type o f succinate d e h y d r o g e n a s e o r w h e t h e r it is c a t a l y z e d by an enzyme distinct from succinate d e h y d r o g e n a s e . A c c o r d i n g to as yet u n p u b l i s h e d results (Oya, H., p e r s o n a l c o m m u n i c a t i o n ) , r e s p i r a t o r y chain c o m p l e x II ( s u c c i n a t e - c o e n z y m e Q reductase) was recently isolated a n d purified f r o m Ascaris muscle m i t o c h o n d r i a . This enzyme system was f o u n d to c o n t a i n f o u r m a j o r and two m i n o r p o l y p e p t i d e s , two of t h e m being similar in m o l e c u l a r weight to the two subunits of m a m m a l i a n succinate d e h y d r o g e -
nase. In addition, Ascaris complex II contains a high amount of a cytochrome, designated cytochrome b55s. The spectral properties of this cytochrome were found similar to the low potential cytochome b fractionating into beef heart respiratory chain complex II. The Ascaris b558 is the major cytochrome constituent of the worm's mitochondrion and was shown to be reduced by N A D H and reoxidized by fumarate in the presence of Ascaris respiratory chain complex I-III. These results together with previous studies [12] suggest that NADH-dependent fumarate reduction in Ascaris and possibly other helminths is brought about by electron transport via respiratory chain complex I (NADH-coenzyme Q reductase), quinone, cytochrome bsss and succinate dehydrogenase operative in reverse (see also Fig. 8). Since in the mammalian pathway hydrogen transfer from N A D H to fumarate does only barely occur, specific requirements not available in the vertebrate mitochondrion must be essential for the parasite to favor the oxidation-reduction in the direction of succinate formation. One of these may concern the nature of the quinone. Many helminths capable of fumarate reduction were found to contain rhodoquinone (Fig. 3) [ 13] instead of the more frequently occurring ubiquinone as a redox carrier mediating electron flow between N A D H dehydrogenation and the fumarate reducing reaction. Previous studies have shown [14] that the redox potential of rhodoquinone (E'o 63 mV) is significantly more negative than that of ubiquinone (E'o = +113 mV), a property which would strongly favor electron transport in the direction of fumarate reduction. Evidence in support of this suggestion has been provided also from studies with bacterial systems [ 15] in which fumarate reduction is mediated by menaquinone (E~o= -74 mV) exhibiting a redox potential considerably lower than that of ubiquinone and the fumarate/succinate couple (Eto = +33 mV). Other factors which may support electron flow from N A D H to fumarate are the properties of the specific polypeptide composition and of the b-type cytochrome demonstrated in Ascaris mitochondrial complex II (see above) and the deficiency of a mitochondrial cytochrome system frequently observed in helminths [12]. In particular the very low activity values of cytochrome c oxidase present in mitochondrial preparations of helminths [ 12] render more difficult the coupling of electron flow from N A D H to molecular oxygen but facilitate the alternative fumarate-linked electron transport pathway. It is interesting to note that the NADH-linked fumarate reduction in helminths allows an uncoupler- and oligomycin-sensitive phosphorylation of ADP similar to that involved in oxidative phosphorylation of the conventional repiratory apparatus [ 12,16]. In this process one ATP per two electrons is conserved at a site comparable to phosphorylation site I. In many helminths succinate, the product offumarate reduction, is a major excretory compound. In contrast, pyruvate, the product of the oxidative branch of the dismutation reaction, is only rarely excreted unchanged. In most cases, this compound is further metabolized to acetyl CoA by oxidation with NAD ÷ as catalyzed by the lipoamide-dependent mitochondrial pyruvate dehydrogenase complex (Fig. 3) [12]. Recently this enzyme has been isolated and purified from Ascaris muscle tissue [ 17]. In
common with the complex isolated from other eukaryotic organisms, this enzyme contains two different subunits and is regulated by reversible phosphorylation. Like in aerobic mitochondria, inactivation of the nematode pyruvate dehydrogenase is stimulated by elevated N A D H / N A D + and acetyl C o A / C o A ratios. Since in tile parasite organelles these ratios arc much higher than m the mitochondria of most aerobic organisms, it was assumed that other factors must account for the high activity of the parasite's pyruvate decarboxylating enzyme. Recently it was suggested that the elevated levels of pyruvate contained in the ascarid mitochondrion may be the important factor in maintaining the enzyme in an active state, even in the presence of highly reduced environmental conditions [18]. In helminths, the product of pyruvate oxidation, acetyl CoA, is primarily metabolized to acetate (Fig. 3), which is in contrast to higher organisms in which this compound is either ultimately oxidized to CO: or used as a precursor for various synthetic purposes. To date the preferred mechanism of free acetate formation iil hetminths is still an open question but it can be suggested that during hydrolysis of acetvl CoA conservation of its "energy-rich' thioester bond may occur through the combined action of an acyl CoA transferase and a thiokinase (eqns. 1 and 21,
Acetyl CoA + succinate ~ acetate + succinyl CoA Succinyl CoA + ADP + P ~ succinate + CoA + ATP
(1) (2)
Direct ATP formation coupled to the cleavage of the thioester bond (eqn. 3), has been demonstrated for protozoan parasites [19,20], but seems unlikely for helminths. Acetyl CoA + A D P + Pi ~ acetate + CoA + ATP
(3)
In general, acyl CoA transfer reactions and acyl CoA-dependent ATP conservation seem to play an important role in the bioenergetics of worm parasites. An example is the process of propionate formation (Fig. 4). A number of reports have indicated [21-23] that this pathway proceeds essentially via reversal of the reactions required for the conversion of propionate to succinate in animal tissues. As expected, this process is associated with ATP synthesis through the coupling o f A D P phosphorylation to the lysis of the carboxyl function of methylmalonyl CoA. Most important is, however, that such an ATP gain can only be achieved if the decarboxylase system is functionally coupled to an acyl CoA transferase which allows the recycling of the "energy-rich' thioester bond of its CoA intermediates. Another area of" awl CoA metabolism which may be of importance for energy generation is the synthesis of 2-methyl branched-chain volatile acids. Such compounds accumulate in a few helminths, and in Ascaris 2-methylbutyrate and 2-methylvalerate are amongst the predominant end products of carbohydrate catabolism. As demontrated in Fig. 5, it seems very likely that these acids arise from the condensation of
Succinate
ADPor GOP~
+ Pi
Propionate ~
~ Succinyl- CoA 5
Succinate'/~'~ ~ . i-'ropionyl-~
Imalonyl-CoA 13 DS"Methyl/~/alOnyl-COA ~ ~--'- ADP÷ Pi ~ , ~ CO2 ATP
Fig. 4. The pathway of propionate formation in helminths (after K6hler et al. [21]).
COSCoA
~H2_CH3~ 1 ~
COSCoA H-CH3 2
COSCoA ~ v I I _.J CoA CH 3 CH3
NADH
COSCoAI CH--CH3 3
COSCoAI C--CH3
w-!-.
CH3
H20 CH3
4
NADH
COSCoA I CH -- CH3
CH3
,~
CoA
COOH I CH-- CH3
CH3
Fig. 5. The pathway of branched-chain volatile fatty acid formation in muscle mitochondria ofA. suurn. T h e example shows the formation of 2-methyl-butyrate from acetyl CoA and propionyl CoA. 1 = acyl CoA transferase; 2 -- 3-ketoacyl CoA reductase; 3 = enoyl CoA hydratase; 4 = enoyl CoA reductase (modified after Komuniecki et al. [25]).
acetyl CoA with propionyl CoA or of two propionyl CoA molecules, with subsequent reduction of the condensation products to the saturated acids [24,25]. A distinctive feature of this biosynthetic pathway is that it constitutes, in principle, a reversal of the I~-oxidation sequence. Like the latter pathway it takes place in the mitochondrial compartment, the acyl intermediates in this process are thioesters of CoA, the reducing power required for substrate reduction is furnished by N A D H and the reaction sequence is dependent on the participation of the mitochondrial electron transport system, as we shall see later. On the other hand, at least some of the enzymes involved in the parasite's branchedchain fatty acid synthesizing pathway are remarkably different from the corresponding 13-oxidation enzymes of mammals. An example is the enzyme catalyzing the N A D H - d e p e n d e n t reduction of 2-methyl-3-ketoacyl CoA derivatives. This enzyme has recently been purified to homogeneity from Ascaris muscle tissue and was shown to be unique in that it reacts overwhelmingly in the direction ofsubstrate reduction and is considerably more active on branched-chain rather than straight-chain acyl CoA substrates [26]. In contrast, the corresponding mammalian 13-oxidation enzyme, 3-
hydroxyacyl CoA dehydrogenase, acts much less preferentially in the reductive direction and is more specific for the straight-chain CoA esters. With the latter enzyme no appreciable activity was demonstrated by employing branched-chain CoA derivatives as substrates [26]. Of particular significance in the fatty acid synthesizing system of Ascaris muscle mitochondria is the penultimate step in which the dehydroacyl CoA compounds are reduced to the saturated CoA esters. Recent studies have indicated [27,28] that the reductive step is dependent on N A D H and proceeds through both a membrane-bound rotenone-sensitive pathway and an entirely soluble enzymatic system (Fig. 6). The soluble component was found to consist of at least two different mitochondrial proteins, an electron-transferring flavoprotein and an enoyl CoA reductase. It is suggested that the membrane-linked pathway serves to transfer reducing equivalents from N A D H , generated from malate and pyruvate oxidation, via a soluble electrontransfer protein to the terminal enoyl CoA reducing enzyme. The sensitivity of the overall reaction ofenoyl CoA reduction towards rotenone would support a pathway in which the respiratory chain-linked N A D H dehydrogenase and rhodoquinone are involved, in a way similar to the pathway o f N A D H - l i n k e d fumarate reduction. Like in the latter system, a most interesting aspect results from the fact that the difference in redox potential between the N A D + / N A D H couple (E'o = -320 mV) and the short-chain enoyl C o A / a c y l CoA ester couples (Ero= - 15 to -30 mV) is sufficiently high to allow an electron transport-associated ATP generation. The recent finding [29] that in Ascaris muscle mitochondria 2-methylvalerate formation was found to be inhibited by 2,4-dinitrophenol, an uncoupler of oxidative phosphorylation, indicates that volatile fatty acid formation may be coupled to ETP. The site of interaction between the rotenonesensitive, m e m b r a n e - b o u n d pathway and the soluble components is, however, as yet unclear. In addition to ETP a second site of energy generation is theoretically feasible in the final step of free branched-chain fatty acid formation (Fig. 5) which occurs with a large
ATP NADH I NADH--DH NAD÷ '
A , RQ
• FR (Succinate
X~
\ Fumarate ETF 2-MB or 2-MV
ECR~--2-MC or 2-MP Fig. 6. A n a e r o b i c electron t r a n s p o r t p a t h w a y s in Ascaris m i t o c h o n d r i a . N A D H - D H = N A D H d e h y d r o g e nase; RQ = r h o d o q u i n o n e ; F R = f u m a r a t e reductase; E T F = electron t r a n s f e r r i n g flavoprotein; E C R = enoyl C o A reductase; 2-MC = 2 - m e t h y l c r o t o n y l C o A ; 2-MP = 2 - m e t h y l p e n t e n o y l C o A ; 2-MB = 2-methylbutyryl C o A ; 2-MV = 2-methylvaleryl C o A ( m o d i f i e d after Rioux and K o m u n i e c k i [29])
drop in free energy upon hydrolysis of the acyl CoA thioester bond. Such a possible ATP formation could proceed either in a single reaction catalyzed by a thiokinase enzyme (analogous to eqn. 3), or by the combined action of two enzymes involving a transferase and a thiokinase enzyme (analogous to eqns. 1 and 2). In summary, the pattern of anaerobic energy generation in volatile fatty acid and succinate forming helminths involves several sites of ATP synthesis within their mitochondrial compartment. As demonstrated in Fig. 7, SLP is associated with methylmalonyl CoA decarboxylation and possibly acyl CoA hydrolysis, while ETP is coupled to NADH-supported reductions of fumarate and presumably enoyl CoA compounds. As outlined in Table II, energy generation in helminths mediated by mixed fermentations and anaerobic electron transport processes would display a surprising advantage over homolactate fermentation. While the latter pathway yields only 2 mol of ATP per mol of glucose catabolized, the overall energy-generating capacity of a helminth oxidizing its carbohydrate to acetate and propionate is more than 5 mol of ATP per mol of glucose. The economic efficiency of these anaerobically functioning energy conserving pathways is, however, still very low compared to that obtained from complete combustion of organic compounds to CO2 and water. From the data shown in Table II it may be also surprising to observe that ATP/glucose ratios are slightly lower when metabolism proceeds until branched-chain acid formation. The explanation for this decrease in efficiency may be the loss in free energy accompanied with the cleavage of the thioester bond during the condensation process of C2 and C3 carbon units to yield the C5 and C6 volatile acids. The synthesis of higher volatile acids in helminths may therefore be not related to the attempt of the organism to improve its energy generation efficiency.
AC
MALATE
I
• PYR
FUMARATE
•AC-CoA.
P SUCC
V ATP
ATP ~-ATP
~" P R O P -
v
ATP
CoA
1
PROP
• 2- MC - CoA
, 2- MP-
ATP
A•
CoA
2-MB-CoA
• 2- MV-
A•
CoA
2-MB
,. 2 -
V
Y
ATP
ATP
MV
Fig. 7. Anaerobic malate-dependent energy conservingmetabolism in Ascaris muscle mitochondria. ATP synthesis was clearly demonstrated for fumarate-linked electron transport and succinate decarboxylation. All other sites of energy conservation indicated in the schemeare still hypothetical. PYR= pyruvate;SUCC = succinate; AC-CoA= acetyl CoA; PROP-CoA= propionyl CoA; AC = acetate; PROP -- propionate; 2-MC-CoA-- 2-methylcrotonylCoA; 2-MP-CoA= 2-methylpentenoylCoA; 2-MB-CoA= 2-methylbutyryl CoA; 2-MV-CoA= 2-methylvalerylCoA; 2-MB= 2-methylbutyrate;2-MV= 2-methylvalerate(modified after Rioux and Komuniecki [29]).
10 TABLE 11 Economic efficiencics of various types of energy conserving metabolic strategies m helminth', Pathway Glucose ~ 3 Glucose ~ 10Glucose ~ Glucose ~
A'[ I:'/gluc~)se 2 lactate 2 acelate g 4 propionate + 2 ('O. 6acetate , 4propionatc ~ 4 MB ÷ MV F Ill('() 6 CO:
2U ~ -. ~iJ 38.1i
Sites of energy conservation used for the above calculations and abbreviations arc indicated in Fig. 7. THE ROLE OF OXYGt N IN THE BIOtLNI-R(IETICSOf: ttt I.MINTttS All worm parasites investigated in vitro were f o u n d to utilize m o l e c u l a r o x y g e n w h e n it is available. There is also some evidence to suggest that this respiration can be coupled to oxidative p h o s p h o r y l a t i o n [12,30]. As in most other eukaryotes, h e h n m t h respiratory systems arc located within their m i t o c h o n d r i a but in contrast to higher a n i m a l s they are frequently mediated by multiple electron t r a n s p o r t chains. In the majority of helminths m i t o c h o n d r i a l respiration may primarily be ascribed to the activity of an electron transport system carrying an alternative oxidase p r o m o t i n g the transfer of electrons to oxygen [8,12[. In contrast to free-living organisms, c y t o c h r o m e c o x i d a s e - d e p e n d e n t respiration often c o n t r i b u t e s to only a m i n o r t~r e'~en negligiblc p o r t i o n of the overall respiration process. U n f o r t u n a t e l y , very little i n f o r m a t i o n is available on the nature o! helminth alternative oxidases and the reason for their occurrence, in preference to or alongside the classical respiratory chain, is still not well understood. Thc a h e r n a t i v e ~xidativc p a t h w a y present in A.scaris muscle m i t o c h o n d r i a has been shown to be tnsensitive to cyanide and a n t i m v c i n A [12,30[ and, il a b r a n c h e d chain is assumed, is likely t,,; emerge from the c o n v e n t i o n a l system at the oxygen side of the N A D H - d e h y d r o g e n a s e segment (Fig. 8). The ascarid oxidase is a p p r o x i m a t e l y 100 times more active than the c y t o c h r o m e oxidase-linked respiration system of the same tissue [12]. In addition, the enzyme is strongly dependent on oxygen tension and accumulates hydrogen peroxide instead of water as the final oxygen reduction product [12,31]. The lact that the alternative oxidase ~d Ascaris m i t o c h o n d r i a is relatively resistant to inhibition bx salicylhydroxamates [ 12], shows that it differs from the cyanide-insensitive alternat lye oxidases f o u n d in w m o u s bacteria and higher plants. In the last few years little progress has been made in our knowledge of the molecula~ assembly a n d f u n c t i o n of h e l m i n t h multiple oxidases. Likewise, the questions as to what extent the various cytochromes d e m o n s t r a t e d in h e l m i n t h m i t o c h o n d r i a arc involved in the different respiration pathways are just b e g i n n i n g to be u n d e r s t o o d Recently, N A D H - c v t o c h r o m e c reductase (complex I-III) was isolated from Ascari.s muscle m i t o c h o n d r i a [32]. As revealed by low temperature difference spectra, this
II
Alternate ] ( ~ 2 2 oxidase Complex
AT P (site 1)
I
02
/
~ , , ~
Succinate Fumarate
Complex ][ Fig. 8. Proposed functional organization of the redox components responsible for electron transport with fumarate and oxygenas terminal acceptors in musclemitochondria 3fA. suum. FP Dand FPs = flavoproteincontaining enzymecomplexesof NADH and succinate dehydrogenase, respectively(modified after K6hler [9]). respiratory chain segment contains two b-type cytochromes, with a-peaks of 559.5 and 563 nm, respectively, and cytochrome ci. Although the enzyme complex was suggested to be as pure as that purified from beef heart mitochondria, its specific activity was more than 20 times less than that of its mammalian counterpart [33]. In the meantime the functional significance of the major cytochrome constituent of the ascarid mitochondrion, cytochrome b558, contained in respiratory-chain complex II, has been investigated to some extent by Oya and his associates (see above). In our laboratory, comparative SDS-PAGE studies on mitochondrial membranes from A s c a r i s muscle and beef heart have revealed remarkable differences in the protein profiles between both systems [11]. As demonstrated in Fig. 9, only very small amounts of protein species similar in molecular weight to the various subunits of beef heart cytochrome c oxidase were found present in the mitochondrial membranes obtained from the parasite. This observation is in complete accordance with the recently reported activity value of A s c a r i s muscle cytochrome c oxidase, being 500 times lower than that of rat liver mitochondria [12]. In addition to the frequently observed low activity o f a phosphorylating cytochrome chain, other pathways resulting in ETP take place in helminth mitochondria. The experimental evidence indicates, however, that these systems are associated with relatively low energy conservation efficiencies [ 12,16,30]. As discussed in the preceding section, the major portion of electron flow observed in helminths producing succinate and volatile fatty acids is directed to organic compounds resulting in ATP synthesis comparable to a site I phosphorylation. When oxygen is available, it may also function as electron acceptor. In A s c a r i s and many other helminths this electron flow to oxygen may be primarily carried by a pathway terminated with an antimycin and cyanide resistant oxidase of unknown identity (Fig. 8).
12
kd
74,5
43
Beeheart f
29
18,5 12
7
li/l
t
Dye front
A$C~FIS muscle
Beef heart Cytochrol~e C oxldase
A
AJ L
Fig. 9. Gel electrophoretic analysis of lhe proteins of mitochondrial membrane preparations from beef heart muscle, Ascaris muscle tissue and purified beef heart cytochrome c oxidase (K6hler, P. and Bachmann, R., unpublished).
From recent experimental work on Ascaris mitochondria [12], indirect evidence supports the assumption that in the alternative pathway, like in fumarate-dependent NADH-oxidation, 1 tool of A T P can be conserved for each pair of electrons transferred to oxygen. No evidence has been obtained with this helminth for an appreciable activity of energy conservation sites II and III. In general, it seems also likely that in the parasite's natural environment under conditions where oxygen concentrations are often limiting the alternative oxidase with its low affinity for oxygen would be only barely operative or switched off. An interesting problem ofhelminth bioenergetics is whether utilization of oxygen by the adult stages will result in an energetic advantage over the anaerobically functioning
13 pathways. In agreement with theoretical considerations and experiments employing mitochondria from Ascaris and F. hepatica [12,30] it may be assumed that, at least in the larger worms, rates of in vitro A T P production are similar under anaerobic and aerobic conditions. There are, however, other species which can use molecular oxygen to significantly increase the overall energy balance. Recent studies by Ramp et al. [34] have shown that phosphorylation activity in extracts obtained from the tiny filarial worm Litomosoides carinii is stimulated by oxygen. This oxygen-supported energy generation was found to occur in addition to that associated with fermentative processes and is most likely due to the function of a conventional type of phosphorylating electron transport system. Calculations made by R a m p and K6hler [35] have shown that the relatively small percentage of substrate flow presumed to be coupled to oxygen-dependent ETP can make a substantial contribution to the overall energy budget of the parasite. A mystery of the filarial worm's respiration is, however, the fact that mitochondrial N A D H oxidase and cytochrome c oxidase activities can account only for less than 10% of the respiration rate observed with intact parasites [34]. Currently the question of whether two different oxygen-consuming principles, a minor mitochondrial one and a major one of unknown location, are present in this organism remains to be answered. A similar situation may exist in Schistosorna rnansoni, the blood-dwelling trematode which was generally believed to be a homolactic fermenter regardless whether oxygen is present or not. From recent results by Van Oordt et al. [36] it can be concluded that a small percentage of glucose catabolized by the adult stage of this worm is completely oxidized to CO2. This process was found to be linked to oxidative phosphorylation and potentially could account for at least one-third of the worm's total aerobic energy production. Other authors have recently examined the relationship between body diameter and type of possible mitochondrial respiratory pathway in various intestinal nematodes of different sizes [37]. As delineated from antimycin A sensitivity, these studies indicate that the portion of a presumed conventional type of respiratory chain correlated well with the body diameter of the worm species. The thinner the organism the greater was the percentage of contribution of an oxygen-consuming pathway sensitive to antimycin A. Very recently, Fry and Beesley [38] have obtained direct evidence for the presence of cytochrome c oxidase in three species of intestinal nematodes. Although the activity of this oxidase showed considerable variation depending upon the worm species and tissue, good correlation was obtained of the distribution of this enzyme with the extent of a mammalian-type electron transport pathway [37]. In contrast to many adult worms, their free-living and parasitic intermediates are often aerobic, containing functional Krebs cycle, 13-oxidation and conventional type of respiration pathways as major catabolic strategies [6,8]. Obviously as a result of an increase in body size, which causes oxygen diffusion problems, and a decreasing access to oxygen during migration of helminths to their ultimate habitats, a steady drop in oxidative capacities often occurs until, in the adult stage, anaerobic strategies become the major ATP-generating pathways.
14
An interesting model for the study of the alterations in bioenergetics occurring during the development of helminths and of the mechanisms initiating the switch from one pathway into another is the liver fluke F. hepatica. Although investigations in this direction are still in their initial phase [7], they have clearly demonstrated that the earl~ liver-parenchymal stage of this parasite has a predominantly aerobic metabolism capable of complete substrate degradation to ( ' O , and water. As shown in Fig. 10, during development ot the flukes, oxidative capacities decline gradually In vitro experiments have shown [7] that in 3-week-old flukes the tricarboxvlate cycle activity is largely suppressed, but concomitantly a pathway resulting in the Iormation ot acetate becomes the predominant oxidative principle for carbohydrate degradation. In this pathway, the major portion of chemical energy may be conserxed by nlilochondrial oxidative phosphorylation. The reducing power necessary to drive respiration m these stages may primarily derive from the single step of pyruvate oxidation to acetate, a pathway which may resemble aerobic acetate formation in L. carinii [34.39]. After arrival of the flukes in their definitive environment, the bile ducts, the organism has to rely almost exclusively on anaerobic redox-processes to meet its energy demands. This metabolism, resulting primarily in the formation of acetate and propionate, follows a pattern essentially identical to that described for other adult hehnmths (Fig. 3). A similar change in oxidative capacities during helminth development ,a,as observed with S. mansoni. Thompson et al. [40] found that 3-h-old schistosomula catabolized glucose to form significant amounts of CO~ and that this CO-, c~olution ~as highly sensitive to cyanide. By 24 h after differentiation from the cercariae COx production from glucose was considerably reduced, while lactate formation was increased. These results suggest that the schistosomulum may continue to rely on respiration-linked energy generation only for a few hours after transformation. 24-h-~4d stages appear t~ be metabolically similar to the adults in that they depend primaril~ on lactate fermen,tation to meet their energy requirements. However, as discussed before, thc adult stage of S. rnansoni may still produce a significant amount of chemical energy through aerobic processes.
1007
. . ~ Complete substrate oxiaalion
-1\
~
60
i
\ /
0
6 12
/ < ! ~ r o b i c acetale""-, formation \~ /
24
50 Fluke age
anaerobic electron
77
114
(days)
Fig. 10. Quantitative relationships between the various energy conserving pathways occurring during tht: development of the liver fluke, F. hepatica (modified after Tielens et al. [7l).
15 Although the developmental changes described appear to be under both genetic and environmental control, there is little evidence on the ways in which the metabolic switches that occur during the life cycle of a helminth are controlled. This central question of how metabolic changes in helminths are regulated remains to be elucidated. SUMMARY From the above discussion it is quite obvious that the bioenergetics in helminths are different in many ways from those found in higher organisms. All adult helminths appear to be able to consume oxygen when it is available but none of them can use it to drive the pathways of complete substrate degradation, like typical aerobic organisms, as a major strategy for energy generation. These properties hold also true for those worms residing in a highly aerobic environment, such as the blood stream or the muscle and lung tissues. Although in a number of recent studies oxygen was found to play apparently a greater role in the bioenergetics of adult helminths than originally thought, energy-generating mechanisms in adult worms seem to place greater emphasis on fermentations and anaerobic electron transport processes. These exhibit relatively low energy conservation efficiencies and result in the formation of a variety of organic end products, most of which must be excreted. The obvious correlation between the type of bioenergetic strategy operative in a particular helminth species and its environmental conditions is not well understood. The increased capacity to generate chemical energy and key metabolites of helminths possessing multiple fermentations and anaerobic respirations may give the organism greater versatility and metabolic flexibility to respond to the environmental changes observed in its corresponding habitat. Other helminths, such as schistosomes and filariids, which have continuous access to a fairly constant nutrient supply, were found to depend primarily on the more inefficiently functioning and primitive strategy of glycolysis for energy production. The reason for the occurrence in helminths of limited oxidative capacities is not completely clear. It may be assumed that the variety of alternative anaerobic pathways have evolved in response to the lack of a circulatory system a n d / o r to the specific, often peculiar, environmental conditions prevailing in most parasitic habitats. An alternative idea put forward by Barrett [8] is that helminth metabolism represents a form of biochemical economy. Most endoparasites have an abundant supply of food and swim as if in a land of Cockain, obviously without any need to extract a maximum amount of chemical energy from the nutrients they take up. On the other hand, the fact that free-living and other larval or juvenile stages of helminths often have a typical aerobic bioenergetic pattern is a clear indication that the D N A of these organisms carries the genetic message for all the enzymes involved in complete substrate degradation. In the adult stages it might be more profitable energywise to maintain many of the genes coding for enzymes involved in terminal oxidation inactivated or repressed
16
and use more simple metabolic strategies rather than complex systems accompanied by an even more complex regulatory machinery for ensuring accuracy of their actions. ACKNOWLEDGEMEN-I S
I wish to thank warmly Drs. H. Oya and R. Komuniecki for providing manuscripts and other communications before publication and Dr. R.P. Casey for a generous gift of beef heart cytochrome c oxidase. 1 am also grateful to Miss E. Lgderach for typing the manuscript and to Mrs. S. Pletscher and Mrs. H. Hug for preparing the figures. REFERENCES
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