antioxidant defense mechanisms in parasitic protozoa

Critical Reviews in Microbiology. 22(4):295-3 14 ( 1 996)

Antioxidant Defense Mechanisms in Parasitic Protozoa

Rajeev K. Mehlotra Division of Geographic Medicine, School of Medicine, W123, Case Western Reserve University, 21 09 Adelbert Road, Cleveland, OH 441 06-4983

ABSTRACT: Many of the parasitic protozoa, such as Entamoeba histolytica, Giardia, Trypa- nosoma, Leishmania, and Plasmodium, are considered to be anaerobes because they can be grown in vitro only under conditions of reduced oxygen tension. However, these parasitic protozoa have been found to be aerotolerant or microaerophilic, and also to consume oxygen to a certain extent. Furthermore, these organisms are highly susceptible to exogenous reactive oxygen species, such as hydrogen peroxide. They must, therefore, detoxify both oxygen and free radical products of enzymatic reactions. However, they lack some or all of the usual antioxidant defense mechanisms present in aerobic or other aerotolerant cells, such as catalase, superoxide dismutase, reduced glutathione, and the glutathione-recycling enzymes glutathione peroxidase and glutathione reductase. Instead, they possess alternative mechanisms for detoxification similar to those known to exist in certain prokaryotes. Although the functional aspects of these alternative mechanisms are yet to be understood completely, they could provide new insights into the biochemical peculiarities of these enigmatic pathogens.

KEY WORDS: antioxidants, parasites, protozoa, Entamoeba histolytica, Giardia, Trypano- soma, Leishmania, Plasmodium.

1. INTRODUCTION

Aerobic organisms produce toxic oxygen derivatives by the reduction of molecular oxygen during aerobic metabolism. The most common free-radical products are the superoxide anion (O;), hydrogen peroxide (H,O,), and their breakdown or metabolic products. Accordingly, they possess elabo- rate defense mechanisms to detoxify the (toxic) products of oxygen reduction. Super- oxide dismutase (SOD) catalyzes the dismutation of 0; to H,O, + 0,. H,O, is converted to H,O + 0, by catalase, peroxidase, and glutathione peroxidase, which uses glutathione (GSH) as the reducing agent. Two major types of SOD carry out the dismutation of 0; in different organisms and

within different organelles and cellular com- partments. These SODS are characterized by the presence of metal ions (either Mn/Fe or Cu and Zn) at the active site of the enzyme, and their sensitivity to cyanide, azide, and H20,. Bacteria contain Mn/Fe- and/or Cu.Zn- SOD, while virtually all eukaryotes contain both types of In most eukaryotes, Mn-SOD activity is restricted to the mitochondria and chloroplast; the enzyme is insensitive to cyanide and H,O,. Fe-SOD has been found in plants and several free-living and parasitic protozoa; the enzyme is inhibited by H,O,, but not by cyanide. Cytosolic sources of 0; are handled in most higher eukaryotes by Cu.Zn-SOD. Recently, the enzyme has also been described from the periplasm of a number of This

1040-84 1 x/96/$.50 Q 1996 by CRC Press, Inc.

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form of SOD is inhibited by both cyanide and H,O,. All of the SODS are inhibited by azide.

Various parasitic protozoa, such as En- tamoeba histolytica, Giardia, trichomonads, Trypanosoma, Leishmania, and Plasmodium, have been shown to be anaerobic in their cultivation requirements. However, metaboli- cally, they have been found to be microaerobic or microaerophilic. They have also been found to produce H,02 as a product of their metabolism in certain cases, and to be highly sensitive to exogenous H,O,. More- over, these parasites, especially the intracel-

lular ones, are naturally exposed to the toxic oxygen metabolites generated by effector immune cells of the host. However, many of them have been reported to lack, or to be extremely deficient in, enzyme systems necessary for the detoxification of H202 (i.e., catalase, glutathione peroxidase) (Table 1). Instead, they possess alternative mechanisms for the detoxification, which have been well characterized from certain prokaryotes (Table 2). The purpose of this review is to provide a broad overview of the strategies, both conventional and alternative, of these parasitic protozoa for the detoxification of oxygen

TABLE 1 Conventional Antioxidant Defense Mechanisms in Various Protozoan Parasites

Parasite

Entamoeba histolytica

Giardia Trypanosoma Leishmania (A) Plasmodium bergheP

P. falciparum Trichomonas vaginalis

Tritrichomonas foetus

Reduced Superoxide glutathionel

Catalase dlsmutase trypanothione

+ + + - + -?

- + +

Glutathionel trypanothlone

reductase

- + + +

+ -?

-?

Glutathionel trypanothione

peroxldase

- +/- +/- +

+ -?

-?

Note: +, present: -, absent; +I-, uncertain: -?, not likely to be present; (A) = Amastigotes. lntraerythrocytic stages.

TABLE 2 Alternative Antioxidant Defense Mechanisms in Certain Protozoan Parasites

NAD(P)H Ascorbate Parasite Cystelne NAD(P)H oxldase peroxldase peroxldase

Entamoeba + + histolytica

Giardia + + Trypanosoma - Trichomonads + +

+

ND ND

+ ND + +!-

ND + Note: +, present; -, absent; +I-, uncertain: ND, not demonstrated.

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metabolites relevant to their survival in their mammalian host. Recent developments and future directions for research in this area are also outlined.

I I . ENTAMUEBA HISTULYTICA

The normal environment of the trophozoites (feeding stages) of E. histolytica is essentially anaerobic, and maintenance of low redox potential is obligatory to its optimal growth in vitro. To maintain a reduced potential in axenic cultures, investigators have used a variety of reducing agents. A combination of L-cysteine (0.1%) and ascorbic acid (0.02%) as the reducing agent was used in the axenic TP-S-1 and TYI-S-33 (or BI-S-33) media.5p6 Good growth of the amoebae in these media was also achieved using L-cysteine (0.2%) without ascorbic a ~ i d , ~ . ~ L-ascorbic acid, D-cysteine, L-cystine, or dithiothreitol: or sodium thioglycollate.'O We have found that GSH (0.2%) supported as good a growth of E. histolytica as the combination of L-cysteine and ascorbic acid, or L-cysteine (0.2%) in BI-S-33 medium." Moreover, 0.25% GSH supported considerably higher growth of amoebae, similar to that obtained with 0.3% L-cysteine."

E. histolytica is an anaerobe. However, the pathogen has also been shown to be oxygen tolerant and to consume oxygen under certain conditions.12 It can tolerate up to 5% oxygen in the gas phase and is able to detoxlfy the products of oxygen reduction in the medium.I3 Although E. histolytica is prima- rily a colonic pathogen, it also invades other organs, mainly the liver. Amoebic liver ab- scess results from pathogenic trophozoites moving from the colon to the liver via the portal circulation. Thus, neither the tissues invaded by the amoebae nor the media used for their cultivation are notably deficient in oxygen. Curiously, E. histolytica lacks catalase,12J4 peroxidase,14 and enzymes of glu-

tathione me tabo l i~m,~~ but Fe-SOD has been found in this p a t h ~ g e n . ' ~ . ~ ~ Hence, if toxic 0; is formed by the partial reduction of oxygen in a variety of aerobic enzymatic oxida- tions, it would be removed with the formation of H,02. How H202 would be detoxified in the absence of catalase is not known. H20, has been shown to be amoebicidal in v i m using an enzymatic H,02-generating systemI7 or in solution.18 It was also found that the addition of catalase to the axenic medium could protect the trophozoites from the lethal effects of H,O2.I8

A point of interest is that the concentration of reducing agent required in the axenic medium (e.g., 0.1% w/v cysteine) is considerably higher than that required for the growth of anaerobic bacteria, in which 0.02 to 0.05% cysteine is adequate and higher concentrations are usually toxic.19 This suggests certain other roles for these reducing agents for the organism, in addition to the maintenance of low redox potential to facilitate growth. Both L-cysteine and L-cystine were found to protect E. histolytica trophozoites from the lethal effects of increased PO, in TYI-S-33 medium.'O The mechanism of protection by L-cystine is obscure, but must be unusual because this amino acid lacks a thiol reducing group. We have proposed that a possible factor effective in detoxifying the H20, might be the availability of "excess" sulfhy- dry1 (-SH) groups.21 This is supported by the observation of Band and Cirrito13 that unin- oculated culture medium containing 0.1% cysteine, exposed to 5% oxygen for 24 h, would support normal growth of amoebae on subsequent inoculation. However, exposure for 72 h was unfavorable for the organism, possibly due to the accumulation of toxic products of oxygen reduction in excess of the ability of the organism to deal with them. The toxicity of the medium could be reversed by adding fresh cysteine, indicating that -SH compounds can detoxify products of oxygen reduction. Cysteine, freshly added

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to the axenic medium,, was found to be rapidly oxidized under conditions used for cul- t ~ r e , ~ and the fact that GSH normally oxidizes less rapidly than cysteine might be important for its better growth-supporting ability. It is also important to mention here that E. histolytica, grown on normal TYI-S-33 medium, has been found to contain significant amounts of glutathione, but only 1/3 of the total was present in the reduced form. How- ever, in glutathione-depleted medium, cysteine was the main thiol component in E. histolytica and was present largely in the thiol

Thus, the reducing agents (thiols) may be utilized for detoxification in E. histolytica, which otherwise has a limited ability to detoxify the products of oxygen reduction.

Reed et al., reported the isolation of an E. histolytica cDNA clone encoding a cysteine-rich 29-kDa protein. The function of the protein is unknown and its localization is not well defined. A database analysis re- vealed substantial sequence homology of the 29-kDa protein to a class of polypeptides found in prokaryotic organisms that may be involved in the inactivation of H202.23 Re- cently, another gene has been identified in E. histolytica encoding a protein (34-kDa) homologous to prokaryotic disulfide oxidoreductases, and it has been suggested that the 29- and 34-kDa proteins represent the two subunits of the active enzyme.24 A more detailed analysis of the protein may help to determine its role in the protection of amoebae against toxic oxygen metabolites. Bruchhaus and TannichZ5 studied the regulation of SOD expression in four E. histolytica isolates under different culture conditions. They found that incubation of amoebae in the presence of an 0,-generating system resulted in an increase in SOD activity between 3.2- and 4.7-fold, as well as an increase in Fe-SOD protein concentration between 2.7- and 5.5-fold. Incubation of amoebae in the presence of a ferrous iron chelator resulted in an increase in SOD

activity between 1.7- and 5.3-fold and in Fe- SOD protein concentration between 2.8- and 7.2-fold. Based on these observations, the authors suggested the possibility that such a regulation of SOD may contribute to protection from toxic oxygen metabolites during tissue invasion by E. histolytica.

111. GIARDIA

Giardia is another anaerobic, amitochondrial protozoan which has been culti- vated axenically in modified TP-S- 1 or TYI- S-33 media with reduced oxygen tension, using L-cysteine and ascorbic acid as the reducing However, growth responses of G. lamblia to reducing agents have been found to be quite different from those observed for E. hi~tolytica.~ In TYI-S- 33 medium, the requirement for reducing agents was found to be quite specific. Only L-cysteine supported the maximum growth of the organism, whereas in TP-S- 1 medium GSH, N-acetyl L-cysteine and mercapto- propionyl glycine also supported 60 to 70% of the maximum growth. L-Ascorbic acid, D- cysteine, and L-cystine were totally ineffec- tive in both the media. However, a combination of L-cystine and ascorbic acid yielded limited growth in TP-S-1 medium.

Giardia does respire and consume oxygen, in that oxygen is actively removed at low partial pressures from culture vessel^.^^^^^ However, the parasite lacks SOD, catalase, peroxidase, and GSH.30.31 L-Cysteine, as in E. histolytica, was found to protect G. lamblia trophozoites under high PO,.~O Although ascorbic acid did not support the growth of the organism, it also protected the trophozoites under high PO,, as did L-cysteine. In another cysteine as well as GSH were found to protect G. lamblia trophozoites from thiol-blocking reactants, indicating a role for the reducing agents for protection of crucial thiol groups.

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Brown et al.30 have found cysteine to be the major low-molecular-weight thiol in Giardia. Low levels of sulfite, thioglycollic acid, and coenzyme A were also observed, while GSH and its intermediates were not detectable. They have also shown that the microaerophilic, amitochondrial parasite Tritrichomonas foetus lacked GSH, but contained high levels of cysteine. Lujan and N a ~ h ~ ~ studied the uptake and metabolism of cysteine by G. lamblia trophozoites. They concluded that L-cysteine is an essential growth factor for Giardia because the parasite (1) cannot take up L-cystine and (2) cannot synthesize cysteine de novo. They have also suggested that the role of cysteine in Giardia may be to protect the organism from the lethal effects of oxygen, as in E. histolytica and Trichomonas vaginalis. 34 Recently, Brown et aL3* found that Giardia contains an alternative mechanism of oxygen detoxification to those seen in higher eukaryotes. High-level NADH-dependent oxidase and low-level NADH-dependent peroxidase activities were detected, similar to anaerobic bacteria. E. histolytica, Tritrichomonas foetus, and Trichomonas vaginalis were also found to contain NADH oxidase activity, confirming earlier observa- t i o n ~ . ~ ~ . ~ ~ However, the authors were unaware of any other reports of an NADH peroxidase being demonstrated in the trichomonads and Entumoeba. Taken together, these enzyme activities, complemented by the high con- tents of endogenous cysteine and thioglycollic acid (a free-radical trap), seem to provide an effective detoxification mechanism in Giardia.

IV. TRYPANOSOMA

Long-term cultivation of pathogenic African trypanosomes in vitro is now well established. To grow trypanosomes axenically, the addition of reducing agents such as

cysteine, 2-mercaptoethanol, or monothio glycerol to the medium was found to bt necessary for continuous c ~ l t i v a t i o n . ~ ~ . ~ ~ Hamm et al.39 found that monothioglycero was the optimal reducing agent for the dif. ferentiation of Trypanosoma brucei trypomastigotes in axenic culture because it sup. ported constant growth rates in all subcultures and was more stable than either cysteine 01 2-mercaptoethanol. Using a cystine-free. minimum essential medium for the axenic cultivation of T. brucei, Duszenko et aL4 found that cell growth was only observed if L-cysteine was either directly added to the medium or was reduced from cystine by the action of reducing agents (monothioglycerol or 2-mercaptoethanol). However, neither reducing. agents alone nor D-cysteine supported cell growth. The need for cysteine was obligatory, despite the fact that methion- ine (a precursor of cysteine biosynthesis) was a constituent of the growth medium. Moreover, T. brucei has been found to effectively transport only cysteine and not cystine.38 These observations led the authors to conclude that cysteine is an essential growth factor for T. brucei and cannot be replaced by other reducing agents, including D-CYS- teine or cystine.

Trypanosoma spp. have been found to generate H202 at fairly high rates and to possess high H202 l e ~ e l s . 4 ~ ~ ~ They have long been thought to lack, or to be extremely deficient in, the enzyme systems necessary for the removal of H,O,. Epimastigotes of T. cruzi have been shown to have a deficient capacity to metabolize H202. They were found to lack catalase and glutathione peroxidase (H20, dependent), but possess SOD44 (Fe-SOD45), GSH, NADPH-dependent glutathione reductase, NADPH oxidase, and a very low activity of ascorbate peroxidase with uncertain function.@ It was subsequently shown that glutathione disulfide reduction in trypanosomatids is dependent upon a unique, low-molecular-weight, thiol-contain-

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ing cofactor (trypanothione), and the NADPH-dependent flavoprotein disulfide reductase (trypanothione reductase) main- tains this metabolite in the dithiol form [T(SH),] within the ell.^,^^ Penketh and Klein4* found that the bloodstream forms of T. b. brucei were not deficient in their ability to metabolize H202, although they were also found to lack catalase, glutathione peroxidase, and ascorbate peroxidase. They were found to metabolize H202 at a significant rate (2.6 nmol/mid108 cells), by a mechanism different from the host. The mechanism involved NADPH and the newly dis- covered cofactor T(SH),. Penketh and Klein48 observed activities of glutathione reductase and NADPH-dependent trypanothione reductase, but a very low activity of trypanothione peroxidase. However, they also found substantial activities of enzymes of the pentose phosphate pathway (e.g., glucose-6-phosphate dehydrogenase, G-6-PDH), which is a prime candidate for the source of NADPH. The authors therefore suggested that NADPH-dependent H202 consumption (reduction of H,02 by NADPH) is the major mechanism involved. Henderson et al.49 identified a trypanothione-dependent peroxidase activity in T. brucei and Crithidia fasciculata and considered that this activity represented the sole mechanism for the removal of H,02 in T. brucei. They also expected similar activities to be present in other species of Try- panosoma and Leishmania that possess the flavoenzyme trypanothione reductase. Penketh et al.50 measured the ability of 12 trypanosomatids to metabolize H202. They confirmed that typical catalase and peroxidase hemoproteins were not important in H202 metabolism, and concluded that the novel process responsible for H,O, metabolism in T. brucei (NADPH-dependent H20, con~umption~~) was common to many, if not all, trypanosomatids.

Carnieri et aL5* confirmed that T. cruzi had lirdted ability to detoxify H202; differ-

ent stages of T. cmzi were found to metabolize low concentrations of H202. The epimastigotes of T. cruzi were found to lack trypanothione peroxidase activity but possess NADPH oxidase and NADPH peroxidase activities. The NADPH peroxidase activity was very low, compared with the true peroxidase activities present in mammalian cells, and was probably due to a nonenzymatic reaction(s) with endogenous T(SH), and/or other thiols. The authors considered that, taking into account the high intracellular concentration of thiols measured, this activity probably accounted for the rates of H20, metabolism detected in intact T. cruzi. Recently, Clark et al.', reported the activity of dehydroascorbate reductase (g1utathione:dehydroascorbate oxidoreduc- tase) in the epimastigotes and trypomastigotes of T. cruzi. The enzyme was known to catalyze a glutathione-dependent reduction of dehydroascorbic acid in plant and animal tissues, but its activity had not been reported in protozoa. They also evaluated the vitamin C content and found that the total vitamin C content (ascorbic acid and dehydroascorbic acid) per milligram of protein was I .6 to 3.6 times higher in trypomastigotes than in epimastigotes. Moreover, trypomastigotes also had a predominance of the reduced form (ascorbic acid, 95%), in contrast to the val- ues found in epimastigotes (24 to 47%). This might be related to the higher resistance/ tolerance to in vitro H20, exposure of trypomastigotes than that of epima~tigotes.~~ The presence of dehydroascorbate reductase activity, along with the ascorbate peroxidase activity,44 suggests that an enzymatic vitamin C redox cycle may also be involved in the scavenging reaction in T. cruzi.

V. LEISHMANIA

The promastigotes of L. donovani and L. tropica are highly susceptible to H202.54.55

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H302 at a concentration of lo4 M was found to inhibit the multiplication of L. donovani promastigotes by 94%, and [3H]thymidine and [3H]uracil incorporation by 95 and 97%, respectively. Continuous generation of H,O, by glucose-glucose oxidase also had a pro- found suppressive effect on promastigote proliferation. However, addition of catalase completely abrogated the growth-inhibitory effect.55 The promastigotes were found to be deficient in catalase and glutathione peroxidase, but possess SOD.54 They readily trigger the macrophage oxidative burst, including the release of H,O,, and 80 to 95% are promptly killed within the cytoplasm of normal resident cells. However, macrophages inherently deficient in the production of oxygen intermediates (577468 cells) exert no appreciable promastigocidal activity.56 Exposure to concanavalin A-stimulated lymphokines effectively enhanced the oxidative response of 577468 cells and, similarly, induced intracellular antileishmanial activity. Inhibiting macrophage H,O, production con- sistently decreased the killing of Leishmania by lymphokine-treated 577468 cells.56

Murray et al.57 found that, although capable of killing the promastigotes of L. donovani, normal mouse peritoneal macrophages exerted no activity against amastigotes. The amastigotes also survived equally well within the in vivo-activated macrophages. However, they could be eradi- cated by in vitro (1ymphokine)-activated cells. The authors concluded that the susceptibility, as well as acquired resistance to L. donovani infection, correlates closely with the host capacity to generate macrophage- activating lymphokines. In the accompanying report, Murray5* demonstrated that, compared with its promastigote predecessor, L. donovani amastigotes contained threefold more catalase and 14-fold more glutathione peroxidase, and were four times more resistant to enzymatically generated H,O,. The intracellular fate of amastigotes was found

to correlate with two particular macrophage properties: (1) the ability to respond to parasite ingestion with oxidative burst activity and (2) the capacity to generate 0; and H,O,. Macrophages capable of simultaneously dis- playing high levels of both the activities (lymphokine-activated cells) killed 90% of amastigotes, whereas those demonstrating neither (577468 cells) permitted amastigotes to replicate. Normal resident macrophages and in vivo-activated cells demonstrated either of the two activities (but not both) and failed to kill amastigotes. Catalase abolished the killing of amastigotes by lymphokine- activated cells, suggesting a primary leishmanicidal role for H202. Extending these observations to human mononuclear phagocytes and examining the interaction of L. donovani with monocytes, Murray and Ca~-tell i~~ demonstrated that the amastigotes were more resistant than promastigotes to killing by mononuclear phagocytes, and that monocytes were considerably more active against both the forms than the monocyte- derived macrophages. The latter finding appeared to reflect the rapid and parallel de- cline in the monocytes oxygen-dependent and -independent antileishmanial mechanisms during (their) in vitro cultivation. The activity of both mechanisms, however, could be effectively restored by lymphokine activation.

In order to determine whether L. donovani survives within macrophages by interfering with the phagocytic oxidative burst or later oxidative responses, Pearson et aL60 examined the interaction of the promastigote and amastigote stages of the parasite with human monocyte-derived macrophages. They concluded that (1) L. donovani does not survive in macrophages by interfering with the phagocytic oxidative burst, (2) does not produce a continuous oxidative activity, and (3) only transiently alters the subsequent macrophage oxidative responses. In a subsequent study, Pearson et al.61 found that the

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survival of L. donovani in human monocytes was dependent on the parasite stage; when promastigotes were ingested, they elicited an oxidative burst, and the majority were killed by oxidative microbicidal mechanisms, whereas amastigotes were ingested and survived to parasitize human monocytes successfully, despite eliciting a phagocytic oxidative burst. In a phagocyte-free system, amastigotes were found to be sevenfold more resistant than were promastigotes to the lethal effects of H,02. Channon et al.62 demonstrated dramatic differences in the binding of, and respiratory burst activity elicited by, promastigotes and amastigotes of L. donovani in murine resident peritoneal macrophages. They explained that, while amastigotes may possess an azide-sensitive mechanism (Fe-SOD, or some other mechanism) that either competes for 0; produced or causes localized inactivation of respiratory burst activity, this cannot account for the full magnitude of the difference between the two forms of the parasite. Transforma- tion and competitive binding studies suggested that the more likely explanation lies in both qualitative and quantitative differences in the distribution of surface ligands involved in binding the parasite to the macrophage membrane; the well-characterized mannose/fucose receptor may be important in the binding of, and respiratory burst activity induced by, promastigotes but not amastigotes6, Channon and BlackwelP3 determined the fate of H202 during its reaction with promastigotes or amastigotes of L. donovani, and also examined the sensi- tivities of both forms of the parasite to reagent or glucose oxidase-generated H202 in a phagocyte-free system. The amastigotes could remove greater amounts of H20, than the promastigotes. It was also observed that, while promastigotes were equally sensitive to either form of H,Oz stress, amastigotes were more resistant to single, larger amounts of reagent H202 than to equivalent amounts

of H202 generated over a 1-h period. These results suggested that the differential responses of promastigotes and amastigotes to different forms of H202 stress may depend upon different H20,-scavenging mechanisms. In the accompanying paper, Channon and Blackwell@ detected catalase in amastigotes but not in promastigotes. Glutathione peroxidase activity was undetectable in either form of the parasite. The total thiol sink (GSH and protein thiols) was greater in promastigotes, but the ability to regenerate GSH via glutathione reductase was equivalent for both forms. Less temperature-dependent, nonenzymatic mechanisms (e.g., an unsaturated lipid sink) also appeared to contribute to the removal of H202 by both forms. The authors, however, supported the idea that the differences in H202 sensitivity between the two forms of the parasite relates to the activity of the direct H,O,-scavenging enzyme, catalase, which appears to operate more efficiently against a bolus of reagent

The trypanothione system appears to be a central feature of antioxidant defense in trypanosomatids. As discussed earlier, protection against H202 has been proposed to involve a trypanothione-dependent enzymatic reac t i~n .~*-~ ' Phenotypes of recombinant L donovani and T. cruzi have been produced that overexpress trypanothione reductase in response to agents that are thought to induce oxidative stress.65 However, the growth of transformed and control cells was equally sensitive to inhibition by these agents. Likewise, growth of both cell types was equally susceptible to inhibition by H202, and control and transformed L. donovani promastigotes metabolized H202 at comparable rates. These experiments suggest that either the reaction normally catalyzed by trypanothione peroxidase is not rate limiting in the clearance of H20, or it is inoperative. Given this, the possibility that a trypanothione-independent system operates for

H202-

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the utilization of H,O, should be considered and warrants further investigation.

The manner in which Leishmania evades intracellular killing is still not completely understood. Indeed, macrophages stimulated with E. coli lipopolysaccharide do secrete large amounts of oxygen metabolites, but they fail to destroy the parasites.& It was thought that Leishmania spp. might interfere with the oxidative metabolism of their host macrophages, and, therefore, the effect of the presence of these intracellular parasites on the respiratory burst of lymphokine-activated and lipopolysaccharide-stimulated mouse macrophages was determined.67 An impairment of the oxidative metabolism of infected vs. noninfected cells was shown; however, the mechanisms of this impairment remained unclear. The parasite has been found to utilize specific receptors (mannose- fucose receptor and the type-three complement receptor CR3) for the attachment and ingestion by r n a c r ~ p h a g e s . ~ ~ - ~ ~ However, the CR3 receptor has been shown to promote phagocytosis but not the generation or release of toxic oxygen intermediates from human phagocy te~ .~~ There is also evidence that the lipophosphoglycan of L. donovani inhibits macrophage protein kinase C activity, an important enzyme that is believed to be responsible for initiating the oxidative b ~ r s t . ~ l - ~ ~ The depletion of protein kinase C rendered macrophages more permissive for the proliferation of L. donovani, suggesting that the inhibition of protein kinase C-dependent events contributes to the survival of this parasite within its host cell. The lipophosphoglycan of L. donovani has also been shown to scavenge potentially cyto- . cidal oxygen metabolites in ~ i t i - 0 . ~ ~ These studies suggest that the lipophosphoglycan of Leishmania also contributes in providing protection against oxidative cytocidal mechanisms of macrophages. Zarley et al.75 investigated the mechanism of H,O, toxicity for L. d. chagasi promastigotes, and factors re-

sponsible for their (relative) H,O, resistance. They found that there were at least two distinct mechanisms for the observed H,O, resistance in different culture forms of promastigotes. First, as promastigotes grow from the logarithmic to the stationary phase in liquid culture medium, they undergo an increase in their virulence for a murine host as well as enhanced resistance to H,02-mediated toxicity. Second, exposure of promastigotes to heat shock also caused them to become more resistant to H,O, toxicity, but through an alternate mechanism. Thus, there are both developmental and inducible factors governing H 2 0 2 resistance in promastigotes, and these likely activate distinct resistance mechanisms. Wilson et al.76 have shown that incubation in sublethal concentrations of H,O, or the redox-cycling agent menadione caused a stress response in promastigotes of L. chagasi. This oxidant- induced response was associated with an increase in the amount of heat-shock protein hsp70. Induction of a stress response by exposure of promastigotes either to heat shock or to sublethal oxidants (H,02 or menadione) caused them to become more resistant to H20, toxicity, and also more virulent to the murine host. Thus, it may be that the adverse environmental changes encountered naturally during the life-cycle of Leishma- nia species (i.e., heat and oxidant) lead to an increase in resistance to the toxic effects of oxidant exposure, which, in turn, may be critical for the survival of parasite in a mammalian host.

VI. PLASMODIUM

Oxidative stress has been defined as any disturbance of the cellular prooxidanthti- oxidant balance in favor of pro oxidant^.^^ Susceptibility to oxidative stress is a well- established feature of malarial parasites.78 The sensitivity of the parasite to oxygen is

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demonstrated by the fact that Plasmodium spp. can be successfully cultured only in atmospheres of low p02.79 H,02 treatment tends to reduce the parasitemia both in vitro and in vivo.80,81 Erythrocytes containing Plasmodium parasites trigger production of reactive oxygen species (ROS) by phagocytes in vitro. The most direct evidence for the killing of parasites by ROS derived from phagocytes has been with P. yoeliiE2 and P. f a l c ipa r~m;~~ detoxification of ROS was shown to prevent the lethal effects of activated phagocytes. Golenser et al.84 also investigated the role of ROS generated by polymorphonuclear leukocytes in the host response against P. falciparum malaria, and concluded that increased oxidative stress induced by polymorphonuclear leukocytes did interfere with the growth of P. falciparum. Finally, the fact that oxidative drugs destroy malarial parasites in v i m and in vivo indicates that Plasmodium spp. are susceptible to oxidative damage.85

Plasmodium spp. have mitochondria and utilize oxygen.8c88 Therefore, ROS-generating processes may be present in the parasite, such as the one-electron reduction of oxygen by the electron transport chain of the mito- chondrion. Deslauriers et al.89 investigated the ability of normal and P. berghei-parasitized erythrocytes to reduce exogenous free radicals in the form of stable nitroxides. Parasitized red cells were found to reduce the lipid-soluble nitroxide probe at a rate that increased with the level of parasitemia. Inhibitors of electron transport increased the rate of reduction. The authors proposed that the reduction occurred via an electron transport chain in the parasite. Production of H202 has been shown in P. berghei,90 but it seems that comparable experiments on other Plas- modium spp. have not been carried out. Hunt and Stocker7 found increased 0,- production in P. vinckei-parasitized erythrocytes. The autooxidation of lipid that occurs in P. vincket9I and P. falciparum-infected red

cells92-94 can be taken as (indirect) evidence for oxidant production by the parasite. 1.n an attempt to verify the biochemical origin of oxidative radicals in malaria-infected erythrocytes, Atamna and Ginsburgg5 found that ROS are produced in the food vacuole of the parasite during the digestion of host cell cytosol, and the H,02 produced during the digestion reaches the host cell compartment. Har-El et al.% suggested that induction of oxidant stress could be mediated by redox- active hemes that are being supplied in an increasing amount by the maturing parasite as it degrades host hemoglobin. They proposed that hemin or hemin-like structures are the appropriate candidates that could catalyze oxidative stress and deregulate the delicate redox balance of the host-parasite system. These s t u d i e ~ ~ ~ ? ~ ~ reestablish the fact that the malarial parasite inflicts an oxidative stress on its host cell, demonstrate the biochemical origin of this stress, and support the idea that heme is involved in the susceptibility of Plasmodium to oxidant stress.

Plasmodium is endowed with its own antioxidant defense mechanisms. Isolated P. berghei parasites were not found to contain endogenous SOD. Host SOD was im- ported and concentrated in the parasites ly- sosomes. In addition, isolated P. berghei also lacked c a t a l a ~ e . ~ ~ , ~ ~ Glutathione reductase has been reported in isolated P. berghei.% In other studies,lOOJO1 isolated P. berghei has been found to contain significant amounts of GSH, glutathione reductase, glutathione peroxidase, and glutathione-S-transferase. Isolated early intraerythrocytic stages of P. falciparum contain catalase and SOD but little or no glutathione peroxidase. However, isolated late-intraerythrocytic stages contain slightly less catalase but significantly more glutathione peroxidase and SOD. P. falciparum, like P. berghei,97 probably acquires most of its SOD from its host because parasite-associated SOD is predominantly cyanide-sensi-

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tive (Cu.Zn-SOD). Unlike P. berghei, however, late stages of P. falciparum contain an additional SOD isozyme that is not cyanide sensitive (Fe- or Mn-SOD), and may represent an endogenous enzyme. Parasites grown in red cells that have been partially depleted of SOD were found to be more sensitive to exogenously generated 03, suggesting some dependence of the parasite on the host SOD.lo2 Ranz and Meshnick103 reported that the endogenous P. falciparum SOD isozyme was most likely Mn-SOD because it was H202 insensitive, and was probably mito- chondrial in origin. Golenser et a1.lW found that this plasmodial SOD existed in various strains of P. falciparum, but the majority of SOD activity found in the parasite was related to the host enzyme. Becuwe et al.Io5 found a cyanide-resistant, H,02-sensitive endogenous Fe-SOD in P. berghei, P. yoelii, and P. vinckei. This endogenous activity represented 25 to 30% of the total SOD activity; the remaining activity may correspond to the internalized RBC Cu.Zn-SOD. The authors suggested that these rodent malarial parasites may be capable of at least partly resist- ing activated oxygen species using an endogenous SOD. Recently, Becuwe et al.06 characterized a cyanide-resistant, H,O,-sensitive endogenous Fe-SOD in P. fakiparum that represented 20 to 30% of the total SOD activity found in the crude extract. The remaining 80% activity may correspond to the RBC SOD, internalized in the digestive system of the parasite. According to these authors, the Fe-SOD activity found in P. falciparum represents the first level of antioxidant defense of the parasite. P. falciparum also has its own glutathione reductase, and it has been speculated that the parasite can store host glutathione reductase during the course of intraerythrocytic maturation.lo7 Recently, the antimalarial activity of a number of glutathione reductase inhibitors was established,lo8 and identification and sequence analysis of the glutathione reduc-

tase gene has been reported.Iw The presence of G-6-PDH in Plasmodium has been a sub- ject of controversy for many years, but it now seems to be convincingly established and partially characterized in P. berghei1I0 and in P. falciparum. Pentose phosphate pathway (or hexose monophosphate shunt, HMS) activity has been measured in intact infected red blood cells.Il2 Recently, Atamna et a l . I l 3 demonstrated considerable HMS activity in free P. falciparum that was about 8 1 to 82% of the total activity seen in parasite- infected red cells. The HMS activity of these infected red cells increased with parasite maturation, which may reflect the oxidative stress generated by the proliferating parasite.

Plasmodium spp. synthesize a number of histidine-rich proteins (HRPs) .~~~ His- tidine analogs have been shown to inhibit the growth of P. falciparum in vitro,117J18 suggesting that HRPs play important role(s) in parasite survival. Histidine is a special agent that can intervene in free-radical reactions in a variety of modes, and can act as a protecting agent.lI9 It has been observed that histidine could prevent P. falcipanun destruc- tion in vitro by freshly isolated and activated human polymorphonuclear neutrophils.120 Based on these observations, it is intriguing to suggest that HRPs may serve an antioxidant function for the parasite.

The malarial parasite derives a number of biochemical advantages from its host erythrocytes, particularly hemoglobin as a source of nutrients and a set of red cell enzymes whose activities can be utilized. From the literature, it seems clear that the parasite produces oxidative radicals that diffuse into the host cell and disturb its redox equilib- rium. Hence, both the hosts and the parasites antioxidant defense systems, must be considerably activated to counteract this oxidative challenge. The energy metabolism of the parasite-infected red cells and its role(s) in maintaining the redox status have been

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reviewed by Ginsburg,l2' Hunt and Stocker,77 and Roth,l12 suggesting, in general, an upregulation of certain key antioxidant mechanisms. Recently, evidence has been presented for the increased uptake and metabolism of riboflavin,122 GSH, vitamin C and vitamin E ~ o n t e n t s , ~ ~ J ~ * ~ ~ and HMS activity' l3 in Plasmodium-infected erythrocytes. Thus, the overall ability of the parasite to withstand oxidant stress in its microenvi- ronment is determined by a number of factors, and seems to be related to the exploita- tion of the erythrocyte antioxidant defense enzymes as well as activatiordproduction of its own protective mechanisms.

VII. CONCLUSIONS AND FUTURE PERSPECTIVES

Careful analysis of the literature indicates the following important characteristics of oxidant-defense systems of parasitic protozoa:

1.

2.

3.

Catalase is absent in Entamoeba histolytica, Giardia, and Trypanosoma. Other metabolic pathways for H202 detoxification exist in these organisms, some of which are similar to those of certain prokaryotes. Cysteine seems to be an essential growth factor for E. histolytica, Giardia, and T. brucei. Giardia and T. brucei cannot transport cystine and cannot synthesize cysteine de novo. Similar studies, however, have not been carried out for E. histolytica. E. histolytica and Giardia (and trichomonads) lack GSH and associated enzymes, but possess high levels of cysteine. Therefore, cysteine-mediated, nonenzymatic reactions seem to function as the detoxification mechanism in these organisms. They have also been stiown to possess alternative pathways

from those described for aerobic eukaryotes. The functional significance of these alternative mechanisms (294 34-kDa protein in E. histolytica, and NADH oxidase and NADH peroxidase in Giardia) has been shown in Salmo- nella typhimurium126 and Haemophilus somnus and Streptococcus faecalis. 127 Trypanosomes need cysteine not only for protein biosynthesis but also for GSH and T(SH), formation, which are present in high amounts. Besides trypanothione metabolism, T. cruzi has been shown to possess an ascorbate peroxidase/dehydroascorbate reductase system as a scavenging mechanism. Ascorbate peroxidase has been found to be effective in scavenging H,O, in various species of cyanobacteria and eukaryotic algae.lZ8 Recently, T. foetus and T. vaginalis were also shown to contain ascorbate peroxidase, which may scavenge low concentrations of Hz02.129 Leishmania has been shown to possess certain nonenzymatic, oxidant-scavenging mechanisms. These include, the surface glycolipid scavenger lipophosphoglycan, and induction of heat-shock proteins in response to heat and oxidant stress. Mycobacterium leprae,74 Salmo- nella typhim~rium'~~ and Escherichia c01i131J32 have already been found to be equipped with such mechanisms. Re- cently, proteoglycans from bovine cornea showed a protective effect on liposome peroxidation induced by Fez+, supporting the hypothesis that proteoglycans may represent part of the antioxidant mechanisms of organisms.133 Plasmodium utilizes a variety of host enzymes for its own protection, although the parasite also has its own defense system. Although a different organism, an analogous situation may exist in Entamoeba histolytica, which

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engulfs bacteria during its commensal phase and RBCs during its invasive phase. It has been hypothesized that the engulfed bacteria and erythrocytes, either intact and/or through their (antioxidant) components, detoxify the products of oxygen reduction to protect the amoebae during i n v a ~ i o n . l ~ ~ ~ ~ How- ever, a possible mechanism of how these components might function within the organism has not been proposed so far.

Beside Leishmania, other species (e.g., E. histolytica, Giardia, Trypanosoma, and Plasmodium) also contain lipophosphoglycan or lipophosphoglycan-like m o l e ~ u l e s . ~ ~ ~ J ~ ~ Similarly, E. histolytica, 139 Giardia, I4O trypa no some^,^^^ and Plasmodi~rn~*-~~ have also been shown to contain heat-shock proteins. However, the functional significance of heat-shock proteins and lipophosphoglycan as antioxidant mechanisms in these organisms has not been explored. It would be interesting to determine whether, as in Leishmania and certain prokaryotes, these molecules provide a (secondary) defense against oxidative stress.

Thus, parasitic protozoa have a diverse arsenal of antioxidant defenses, and many of these are shared with the prokaryotes. Re- cent developments in cloning specific genes, and the isolation of regulatory mutants have helped further our understanding of the regulation of these defense mechanisms in prokaryotes. Analogous studies among the parasitic protozoa hold the promise of being similarly fruitful.

ACKNOWLEDGMENTS

My sincere thanks are due to Dr. Eric Pearlman, Assistant Professor, Division of Geographic Medicine and Department of Ophthalmology, for his constant support and encouragement, and his valuable coopera-

tion and help in various ways in completing this review. I also want to recognize the contribution of Dr. Wes Kroeze of the De- partment of Biochemistry, who has read the manuscript and contributed valuable sugges- tions and criticisms. Without such help, this review would indeed be poorer.

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