Entamoeba histolytica lacks trypanothione metabolism

Molecular and Biochemical Parasitology 103 (1999) 61 – 69 www.elsevier.com/locate/parasitology

Entamoeba histolytica lacks trypanothione metabolism Mark R. Ariyanayagam, Alan H. Fairlamb * Department of Biochemistry, Wellcome Trust Building, Uni6ersity of Dundee, Dundee DD1 5EH, Scotland, UK Received 25 March 1999; received in revised form 4 June 1999; accepted 9 June 1999

Abstract Entamoeba histolytica lacks glutathione reductase activity and the ability to synthesise glutathione de novo. However, a recent report suggested that exogenous glutathione can be taken up and conjugated to spermidine to form trypanothione, a metabolite found so far only in trypanosomatids. Given the therapeutic implications of this observation, we have carefully analysed E. histolytica for evidence of trypanothione metabolism. Using a sensitive fluorescence-based HPLC detection system we could confirm previous reports that cysteine and hydrogen sulphide are the principal low molecular mass thiols. However, we were unable to detect trypanothione or its precursor N 1-glutathionylspermidine [ B0.01 nmol (106 cells) − 1 or B1.7 mM]. In contrast, Trypanosoma cruzi epimastigotes (grown in a polyamine-supplemented medium) and Leishmania dono6ani promastigotes contained intracellular concentrations of trypanothione two to three orders of magnitude greater than the limits of detection. Likewise, trypanothione reductase activity was not detectable in E. histolytica [ B 0.003 U (mg protein) − 1] and therefore at least 100-fold less than trypanosomatids. Moreover, although E. histolytica were found to contain trace amounts of glutathione (approximately 20 mM), glutathione reductase activity was below the limits of detection [B 0.005 U (mg protein) − 1]. These findings argue against the existence of trypanothione metabolism in E. histolytica. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Thiols; Trypanothione; Glutathione; Entamoeba histolytica; Evolution; Mitochondrion

1. Introduction All eukaryotic organisms require defences against oxidant stress caused by reactive oxygen intermediates arising from cellular metabolism in an aerobic environment. Important amongst these defences are low molecular mass thiols such as * Corresponding author. Tel.: +44-1382-345155; fax +441382-345542. E-mail address: [email protected] (A.H. Fairlamb)

glutathione and associated cycling enzymes such as glutathione reductase and glutathione peroxidase [1]. In contrast to aerobic mitochondrial protozoans such as trypanosomatids or Plasmodium [2], amitochondrial protozoans such as Entamoeba histolytica, Giardia and Trichomonas apparently lack glutathione metabolism [3,4] and instead contain cysteine as their principal lowmolecular mass thiol [5,6]. However, this widely accepted view has recently been challenged by Ondarza et al. [7] who claim to have detected trypanothione [N 1,N 8bis(glutathionyl)spermidine]

0166-6851/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 6 - 6 8 5 1 ( 9 9 ) 0 0 1 1 8 - 8

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in E. histolytica, a redox metabolite previously thought to be unique to trypanosomatids [8,9]. Given the importance of trypanothione metabolism as a validated therapeutic target in Leishmania [10–12], we decided to further investigate thiol metabolism in E. histolytica in the hope of extending our attempts at drug discovery to include this medically important parasite. Our studies presented here suggest that trypanothione metabolism is absent or quantitatively insignificant in this organism.

freeze-thawing three times prior to sonication to abolish infectivity. A neutralised acid extract of E. histolytica HK9 cells (zymodeme II) was prepared and supplied by Dr R. Ondarza (Instituto Nacional de Salud Publica, Cuernavaca, Mexico). The cells (550 mg wet weight) were harvested at 36 h, extracted with 10% perchloric acid, neutralised with potassium hydroxide and centrifuged (10 000× g, 15 min, 4°C) to yield 4 ml of extract.

2.3. Enzyme assays 2. Methods and materials

2.1. Culture Cultures of E. histolytica strain HM-1: IMSS, clone 2 (zymodeme II) were initiated with a 1 ml inoculum in 8 ml YI-S medium and passaged every 5–6 days [13]. Trypanosoma cruzi X10 clone 6 (MHOM/BR/78/Silvio) epimastigotes were cultured in RTH/FCS medium supplemented with 5 mM putrescine [14]. Leishmania dono6ani LV9 (MHOM/ET/67-/HU3) promastigotes were cultured in GIM/FCS medium as described previously [15].

2.2. Cell lysate preparation E. histolytica cells (8×106) from 3-day-old cultures were pelleted by centrifugation (375× g, 10 min, 4°C) and lysed in 0.4 ml 20 mM potassium phosphate pH 7.2 containing 1 mM EDTA, 5 mM benzamidine, 5 mM phenanthroline, 10 mM (2S,3S) - trans - epoxysuccinyl - L - leucylamido - 3methylbutane (E64c) and 1 mM dithiothreitol. Cells were sonicated with four 30-s pulses with intermittent cooling on ice followed by centrifugation to remove cellular debris (10 000× g, 15 min, 4°C). The resulting supernatant was subjected to ultracentrifugation at 50 000×g for 45 min at 4°C followed by dialysis in a Pierce dialysis cassette (Pierce Warriner Ltd, UK) against the lysis buffer ( × 250 vol. with one change). Similar ultrafiltrates were prepared from T. cruzi epimastigotes and L. dono6ani promastigotes (approximately 1×109 cells, late log phase) with

Trypanothione and glutathione reductase activities were assayed spectrophotometrically at 27°C by monitoring the oxidation of NADPH at 340 nm as described previously [15]. NADPH or NADH: flavin oxidoreductase activities were assayed as described [16]. One unit of activity (U) is defined as the amount of enzyme required to catalyse the conversion of 1 mmol NADPH to NADP + (or NADH to NAD + ) min − 1 at 27°C. Protein concentrations were determined by the Bradford method [17] using bovine serum albumin as standard.

2.4. Thiol analysis Freshly harvested cells (4–6×106 E. histolytica cells, 5×107 L. dono6ani promastigotes or 1–2× 108 T. cruzi epimastigotes) were derivatised with monobromobimane [18] and thiols analysed by HPLC [19] with the following modification to the separation conditions. In order to resolve the cysteine bimane derivative from the bimane reagent peak, the analytical system for the separation of amino compounds was used [19] in which linear gradients of 0–20% Solvent B over 60 min, followed by 20–75% Solvent B over 40 min were applied after sample injection. Some freshly harvested E. histolytica cells were washed with icecold phosphate-buffered saline (Na + 162.95 mM, PO34 − 10 mM, Cl − 145.45 mM, pH 7.18) prior to derivatisation. Aliquots (50 ml) of the neutralised acid extract supplied by Dr Ondarza were derivatised after reducing thiols enzymatically with 0.2 U ml − 1 recombinant trypanothione reductase [20], 8.5 mM

M.R. Ariyanayagam, A.H. Fairlamb / Molecular and Biochemical Parasitology 103 (1999) 61–69

trypanothione disulphide and 375 mM NADPH in 20 mM HEPPS buffer, 2 mM diethylenetriaminepentaacetic acid (DTPA), pH 8.0 in a total volume of 100 ml. After incubation at 28°C for 30 min, reactions were derivatised with 50 ml of 2 mM monobromobimane followed by the addition of 150 ml 4 M (Li + ) methanesulphonate, pH 1.6. Some samples were treated with 50 ml of 10 mM N-ethylmaleimide prior to monobromobimane derivatisation. Authentic thiol standards were prepared by reduction of disulphides either enzymatically with trypanothione reductase and required co-factors [21] or chemically with dithiothreitol [22], followed by derivatisation with monobromobimane and purification by preparative HPLC.

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prised of this bimane product. Authentic standards of sulphite and thiosulphate co-eluted with U1 and U2, respectively. It should be noted that thiosulphate does not react with N-ethyl maleimide [22] and therefore thiosulphate would still react with monobromobimane to form a fluorescent product. U2 can therefore be tentatively assigned as thiosulphate. Since the size of reagent peak R1 in Fig. 1 indicates that bimane reagent is in vast excess over thiols, such that the reagent peaks obscures

2.5. Reagents Trypanothione was purchased from Bachem (UK) Ltd. Syn-(methyl,methyl)bimane was a kind gift from Professor E. Kosower (University of Tel-Aviv, Israel). Sources of other reagents were as described previously [18,21].

3. Results In a preliminary study, fresh E. histolytica cells from 3-day-old cultures were analysed for thiol content by derivatisation with monobromobimane. As a control, an equivalent number of cells were treated with N-ethyl maleimide prior to derivatisation. Since most thiols react with Nethyl maleimide to form derivatives that are unreactive with monobromobimane, comparison of both chromatographic traces in Fig. 1 gives an indication of whether any particular peak is due to a fluorescent thiol or a fluorescent non-thiol component. In the sample pre-treated with Nethyl maleimide (trace B), fluorescent peaks U1, U3, U4 and U6 were abolished completely whilst the peak area of U5 decreased by approximately 50%. The peak area of U2 increased by approximately 40% whilst the main reagent peaks labelled R1 (due to monobromobimane), R2 and R3 remained after N-ethyl maleimide treatment. Authentic syn-(methyl,methyl)bimane co-eluted with U5, suggesting that U5 may be partially com-

Fig. 1. HPLC chromatograms of freshly derivatised E. histolytica HM-1 thiols. Trace (A): solid line, cells (6.9×105) derivatised with monobromobimane; broken line, syn(methyl,methyl)bimane, 100 pmol; trace (B): solid line, cells (4.1 × 105) derivatised with N-ethylmaleimide prior to monobromobimane treatment; broken line, reagent blank treated with N-ethylmaleimide prior to monobromobimane derivatisation; U1 – U6 indicate peaks that are either abolished or change significantly upon N-ethylmaleimide treatment, R1 – R3 indicate reagent peaks. Equivalents of 3.45 × 104 cells were injected for both E. histolytica samples.

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some of the unknown thiols, more E. histolytica cells were used to accurately quantify thiol content in subsequent experiments. Approximately 4 – 6 × 106 cells were used for each derivatisation which is equivalent to the number of cells used (5× 106) in a previous study [3]. As shown in Fig. 2 (traces A and D), U3 co-elutes with cysteine, U4 co-elutes with glutathione and U6 co-elutes with hydrogen sulphide. In addition, spiking cell samples with authentic glutathione (trace B) and hydrogen sulphide (trace C) produces corresponding increases in the peak areas of U4 and U6, respectively, thus confirming that U4 and U6 have the same chromatographic profiles as glutathione and hydrogen sulphide, respectively. In contrast, when spiked with authentic trypanothione and glutathionylspermidine (trace B) none of the other peaks showed any corresponding increase in peak area. A clear separation between the trypanothione peak and hydrogen sulphide peak is evident in trace (B). Cells derivatised with only a slight excess of monobromobimane did not show appreciable sulphite or thiosulphate peaks (Figs. 1 and 2). None of the peaks co-eluted with authentic bimane standards of ovothiol A (51 min), g-glutamylcysteine (56 min), ergothioneine (58 min) or cysteinylglycine (59 min) (not shown). Thus the major thiols in E. histolytica can be assigned as cysteine and hydrogen sulphide in agreement with previous data [3]. The cysteine content (2.85 nmol (106 cells − 1), Table 1) of E. histolytica shown is somewhat lower than those reported by Fahey et al. (5.1 nmol (106 cells − 1)) [3], possibly due to the different media used for cell growth (YI-S and TYI-S33, respectively). Cells washed in phosphatebuffered saline prior to bimane treatment have a decreased thiol content, particularly cysteine (40% of unwashed cells) possibly due to trace amounts of medium contaminating the cell pellet. Comparison of E. histolytica with representative trypanosomatids illustrates the striking difference in the intracellular concentrations of the major thiols present. T. cruzi epimastigotes and L. dono6ani promastigotes have respectively, 21- and 170-fold higher concentrations of glutathione than E. histolytica. Trypanothione and glutathionylspermidine, which are readily detectable in

Fig. 2. HPLC chromatograms of freshly derivatised E. histolytica HM-1 thiols. Trace (A): cells (5.44 × 106) derivatised with monobromobimane; trace (B): sample spiked with prederivatised glutathionyl conjugate standards, spike contained 30 pmol glutathione, 70 pmol glutathionylspermidine and 60 pmol trypanothione; trace (C) sample spiked with 140 pmol pre-derivatised hydrogen sulphide (H2S); trace (E) pre-derivatised standard containing 200 pmol cysteine (CYS), 100 pmol glutathione (GSH), 105 pmol glutathionylspermidine (GspdSH), 95 pmol trypanothione [T(SH)2] and 224 pmol of H2S. Equivalents of 2.72 × 105 cells were injected for traces A – C.

trypanosomatids by our analytical method, were not detectable in E. histolytica (B 0.01 nmol (106 cells) − 1 or B1.7 mM). The total intracellular thiol concentration of E. histolytica is 5- and 60-fold lower than T. cruzi and L. dono6ani, re-

M.R. Ariyanayagam, A.H. Fairlamb / Molecular and Biochemical Parasitology 103 (1999) 61–69

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Table 1 Thiol content of E. histolytica compared with T. cruzi and L. dono6ani a Cell type

Thiol Cys

Thiol content, nmol (10 6 cells)−1 E. histolytica HM-1 (un2.99 0.4 washed) E. histolytica HM-1 (washed) 1.19

Total SH groups OSHb

GSH

GspdSH

H2S

T(SH)2

B0.08c

0.12 9 0.01

B0.01c

1.09 9 0.19

B0.01c

4.06

B0.08

0.11

B0.01

0.79

B0.01

2.09

B1.7 44 914 492 950

134 B40c B40

B1.7 360 524 9 7 1800 53609 700 21700

Intracellular concentration (mM) E. histolytica HM-1 (washed) 202 B13.6 19 T. cruzi X10-6 1679 9 1339 13 4079 21 L. dono6ani LV9 22809 310 49709 525 32709 300 d

a E. histolytica trophozoites were harvested at 3 days growth and trypanosomatids were harvested at late log phase. Cells were derivatised with monobromobimane as described in methods and materials section. Values are the means ( 9standard error of the mean) of triplicate cultures, except for E. histolytica cells that were washed with phosphate-buffered saline which are the average of duplicate cultures. b OSH, ovothiol; other abbreviations as described in legend to Fig. 2. c Values are below the limits of detection. d Intracellular concentrations calculated using a cell volume of 5.9 ml (106 cells)−1 for E. histolytica [33], 5.5 ml (108 cells)−1 for T. cruzi [21] and 1.2 ml (108 cells)−1 for L. dono6ani [34].

spectively. Of this, 83 and 67% are found in glutathione and glutathionylspermidine conjugates in T. cruzi and L. dono6ani, respectively, whilst only 5% is found as glutathione in E. histolytica. Trypanothione reductase activities in ultrafiltrates of E. histolytica were below the limits of detection (B0.003 U (mg protein) − 1) in contrast to extracts of T. cruzi epimastigotes and L. dono6ani promastigotes prepared under identical conditions (Table 2). In agreement with Fahey and Newton [3], we could not detect any significant glutathione reductase activity in E. histolytica ( B 0.005 U (mg protein) − 1). The lack of glutathione reductase or trypanothione reductase activity cannot be ascribed to inadequate extraction or inactivation due to proteolysis since we could detect NADPH:flavin oxidoreductase activity (0.34 U (mg protein) − 1) comparable to that reported previously (0.10 U (mg protein) − 1) [16]. This report also stated that there is approximately 5% activity with NADH as with NADPH. Our value was 5.3% of that obtained for NADPH. These results suggest that the central components of trypanothione metabolism (trypanothione, glutathionyl-

spermidine and trypanothione reductase) are absent from E. histolytica. In order to try to resolve our negative findings with the recent report indicating the presence of trypanothione in E. histolytica [7] we obtained a cell extract prepared by the authors of this study. Fig. 3 (trace A) shows the chromatogram of such a bimane-treated neutralised acid extract of E. histolytica following bimane treatment. Small peaks are visible where bimane adducts of cysteine, sulphite, thiosulphate and hydrogen sulphide elute, but no glutathione or trypanothione peaks are present. In addition, a large unidentified peak labelled as U7 is visible. After reducing the extract enzymatically with recombinant trypanothione reductase and NADPH, no appreciable increase in any peak area was noted (trace B). However, following incubation with trypanothione reductase, NADPH and exogenous trypanothione, a trypanothione peak is now visible and clearly distinct from hydrogen sulphide (trace C). The fact that 91% of the trypanothione added as the disulphide is recovered as dihydrotrypanothione indicates that the acid extracts do not contain inhibitors of the enzymatic reducing system.

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Table 2 Enzyme activities of E. histolytica, T. cruzi and L. dono6ani ultrafiltrates Cell type

E. histolytica HM-1 T. cruzi X10-6 L. dono6ani LV9

Enzyme activity in ultrafiltratea (U mg protein−1) TR

GR

F/NADPH

F/NADH

B0.003 0.099 0.01 0.349 0.03

B0.005 –b –

0.34 90.02 – –

0.02 – –

a TR, trypanothione reductase; GR, glutathione reductase; F/NADPH, NADPH:flavin oxidoreductase; F/NADH, NADH:flavin oxidoreductase. Assays were done in triplicate apart from F/NADH assay which was done in duplicate. b A dash indicates value not determined.

The peaks which co-elute with sulphite, thiosulphate, cysteine, glutathione, U5 and hydrogen sulphide are all increased due to reduction via thiol-disulphide exchange with the exogenously added trypanothione/trypanothione reductase system. Upon treatment of samples A – C with Nethyl maleimide prior to bimane derivatisation (traces E–G), sulphite, cysteine, glutathione, trypanothione and hydrogen sulphide are completely abolished. The size of the U5 peak is considerably decreased, suggesting that a non-thiol component possibly co-elutes with syn-(methyl,methyl)bimane at the position of U5. U2 (thiosulphate, cf., Fig. 3, traces C and G) and U7 (cf., Fig. 3, traces A–C with E – G) were unchanged by Nethyl maleimide treatment. Since U7 was not present in freshly derivatised cells (Figs. 1 and 2) and did not appear in our reagent blanks, it may be the product of an unknown component present in the acid cell extract. Reduction of disulphides in the extract using NADPH, glutathione and glutathione reductase gave similar results (traces not shown). The previous study [7] reporting the presence of trypanothione employed dithiothreitol as reducing agent. The extreme difficulty in assigning peaks when reducing cell extracts with dithiothreitol is illustrated in Fig. 4. Although cysteine and glutathione peaks are readily detectable after reduction of the sample with dithiothreitol (trace A), the region where trypanothione and hydrogen sulphide elute are obscured by the bisbimane adduct of dithiothreitol eluting at 82 min and two additional peaks D1 and D2 that are present in the dithiothreitol blank (trace B). In other experi-

ments where dithiothreitol was added in excess over monobromobimane, we observed additional peaks that elute near the positions of U5 and U7 which we attribute to mono-bimane adducts of dithiothreitol (data not shown). Clearly, reduction of disulphides using dithiothreitol is best avoided when analysing thiol content by fluorescencebased methods.

4. Discussion The data presented here suggest that trypanothione metabolism is absent from E. histolytica. Trypanothione and glutathionylspermidine were below the limits of detection of our sensitive HPLC system under conditions, which readily quantify these thiols in T. cruzi and L. dono6ani. If trypanothione is indeed present in E. histolytica then it is present at concentrations two to three orders of magnitude lower than in the trypanosomatids. Similarly the absence of significant trypanothione reductase activity in E. histolytica (B 0.003 U (mg protein) − 1) argues against the presence of trypanothione metabolism. Likewise, although measurable quantities of glutathione could be detected in cells grown in undefined medium, glutathione reductase activity is below the limits of detection. YI-S medium contains yeast extract that is rich in glutathione and could be taken up via endocytosis as previously suggested [3]. Quantitatively, glutathione content represents 5% of the total measured intracellular thiols, with cysteine and hydrogen sulphide representing the major components. Two of the other

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Fig. 3. HPLC chromatograms of E. histolytica HK-9 thiols from a neutralised acid extract. Trace (A): extract derivatised with monobromobimane; trace (B): extract derivatised with monobromobimane after incubation with exogenous trypanothione reductase and NADPH; trace (C): extract derivatised with monobromobimane after incubation with exogenous trypanothione, trypanothione reductase and NADPH; trace (D): pre-derivatised standard containing 200 pmol cysteine (CYS), 100 pmol glutathione (GSH), 105 pmol glutathionylspermidine (GspdSH), 95 pmol trypanothione [T(SH)2] and 224 pmol of H2S; trace (E): extract from (A) treated with N-ethylmaleimide prior to monobromobimane treatment; trace (F): extract from (B) treated with N-ethylmaleimide prior to monobromobimane treatment; trace (G): extract from (C) treated with N-ethylmaleimide prior to monobromobimane treatment; trace (H) reagent blank treated with N-ethylmaleimide with monobromobimane derivatisation. U7 indicates an unknown peak in extract, formed after derivatisation with monobromobimane. For all samples, equal amounts of extract (50 ml, equivalent to 6.9 mg wet weight cells) were derivatised and then injected (equivalents of 0.23 mg wet weight cells) for HPLC elution.

unidentified minor components can be tentatively assigned as sulphite and thiosulphate. One other dominant peak is U5, which coelutes with syn-(methyl,methyl)bimane. Fahey and Newton have noted that monobromobimane can serve as an electron acceptor as well as undergoing nucleophilic substitution reactions with thiols and other nucleophiles [23]. These authors noted that constituents in cell extracts, especially from photosynthetic organisms, can reduce monobromobimane to syn-(methyl,methyl)bimane and that this reaction can be blocked by N-ethyl maleimide to varying degrees, depending on the sample. Thus it is impossible to conclude from these studies whether or not the U5 peak contains an additional unknown thiol.

All of the data presented here are both quantitatively and qualitatively in agreement with those of Newton et al. [3] and in stark contradiction to the report by Ondarza et al. [7]. Owing to the different separation and analytical techniques it is rather difficult to resolve this discrepancy. Certainly this cannot be ascribed to the use of different lines of E. histolytica or different culture conditions since we were unable to detect trypanothione and glutathionylspermidine in extracts kindly provided by Dr R. Ondarza. These authors estimate both metabolites to be approximately 0.1 nmol (106 cells) − 1 [7] which should therefore be readily detectable like glutathione (0.11 nmol [106 cells] − 1) using our analytical system. One possibility is that minor contaminants that co-elute

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with trypanothione or glutathionylspermidine in the dithiothreitol used to reduce their preparations have been erroneously purified (cf., peak D1, Fig. 4). During evolution, glutathione metabolism is proposed to have been acquired by eukaryotes from endosymbionts that also gave rise to mitochondria and consequently aerobic metabolism [24 – 26]. Indeed, the absence of mitochondria and glutathione metabolism in E. histolytica has been

cited in support of this hypothesis [3,27]. However, there is now strong molecular and biochemical evidence that E. histolytica has acquired, but subsequently lost, its mitochondrial function [28]. Certain genes such as pyridine nucleotide transhydrogenase and chaperone cpn 60 which are thought to have originated from endosymbionts are postulated to have been transferred to the nuclear genome and subsequently retained [28– 30]. No mitochondrial genome encodes any enzymes of glutathione metabolism [24] and a similar genetic transfer to the nuclear genome is postulated to have taken place [28,29]. Presumably, reversion to anaerobic metabolism in a microaerophilic environment did not favour retention of glutathione as the key anti-oxidant system in E. histolytica. Two other amoebal enzymes, Eh34 (E. histolytica NADPH: flavin oxidoreductase) [31] and Eh29 (E. histolytica alkyl hydroperoxide reductase, a thiol specific anti-oxidant enzyme like protein) [32] may have subsumed the roles of glutathione and trypanothione systems present in other eukaryotes.

Acknowledgements We are grateful to Drs Jorge Tovar and Graham Clark (LSHTM, London, UK) for kindly providing fresh E. histolytica cultures. We also thank Dr Raul Ondarza for providing E. histolytica extracts and communicating his findings to us. This work was supported by the Wellcome Trust.

References

Fig. 4. HPLC chromatograms of E. histolytica HK-9 thiols from a neutralised acid extract. Trace (A): extract (50 ml) reduced with dithiothreitol prior to monobromobimane derivatisation, dithiothreitol is in excess of monobromobimane in a ratio of 2:1, an equivalent of 0.46 mg wet weight cells was injected; Trace (B): dithiothreitol (1000 pmol) derivatised with monobromobimane, dithiothreitol to monobromobimane ratio is 2:1; Trace (C): pre-derivatised standard (amounts as in Figs. 2 and 3). D1, D2 indicate additional peaks formed upon derivatisation with dithiothreitol present, bisDTT indicates bisbimane adduct of dithiothreitol.

[1] Meister A, Anderson ME. Glutathione. Annu Rev Biochem 1983;52:711 – 60. [2] Fairlamb AH. Novel biochemical pathways in parasitic protozoa. Parasitology 1989;99S:93 – 112. [3] Fahey RC, Newton GL, Arrick B, Overdank-Bogart T, Aley SB. Entamoeba histolytica: a eukaryote without glutathione metabolism. Science 1984;224:70 – 2. [4] Brown DM, Upcroft JA, Edwards MR, Upcroft P. Anaerobic bacterial metabolism in the ancient eukaryote Giardia duodenalis. Int J Parasitol 1998;28:149 – 64. [5] McLaughlin J, Aley S. The biochemistry and functional morphology of the Entamoeba. J Protozool 1985;32:221 – 40.

M.R. Ariyanayagam, A.H. Fairlamb / Molecular and Biochemical Parasitology 103 (1999) 61–69 [6] Brown DM, Upcroft JA, Upcroft P. Cysteine is the major low-molecular weight thiol in Giardia duodenalis. Mol Biochem Parasitol 1993;61:155–8. [7] Ondarza RN, Tamayo EM, Hurtado G, Hernandez E, Iturbe A. Isolation and purification of glutathionyl-spermidine and trypanothione from Entamoeba histolytica. Arch Med Res 1997;28:S73–5. [8] Fairlamb AH, Blackburn P, Ulrich P, Chait BT, Cerami A. Trypanothione: a novel bis(glutathionyl)spermidine cofactor for glutathione reductase in trypanosomatids. Science 1985;227:1485–7. [9] Fairlamb AH, Cerami A. Metabolism and functions of trypanothione in the Kinetoplastida. Annu Rev Microbiol 1992;46:695 – 729. [10] Tovar J, Wilkinson S, Mottram JC, Fairlamb AH. Evidence that trypanothione reductase is an essential enzyme in Leishmania by targeted replacement of the tryA gene locus. Mol Microbiol 1998;29:653–60. [11] Tovar J, Cunningham ML, Smith AC, Croft SL, Fairlamb AH. Down-regulation of Leishmania dono6ani trypanothione reductase by heterologous expression of a trans-dominant mutant homologue: effect on parasite intracellular survival. Proc Natl Acad Sci USA 1998;95:5311 – 6. [12] Dumas C, Ouellette M, Tovar J, Cunningham ML, Fairlamb AH, Tamar S, Olivier M, Papadopoulou B. Disruption of the trypanothione reductase gene of Leishmania decreases its ability to survive oxidative stress in macrophages. EMBO J 1997;16:2590–8. [13] Diamond LS, Clark CG, Cunnick CC. YI-S, a casein-free medium for axenic cultivation of Entamoeba histolytica, related Entamoeba, Giardia intestinalis and Trichomonas 6aginalis. J Euk Microbiol 1995;42:277–8. [14] Pereira ME, Hoff R. Heterogeneous distribution of neuraminidase activity in strains and clones of Trypanosoma cruzi and its possible association with parasite myotropism. Mol Biochem Parasitol 1986;20:183–9. [15] Cunningham ML, Fairlamb AH. Trypanothione reductase from Leishmania dono6ani—purification, characterisation and inhibition by trivalent antimonials. Eur J Biochem 1995;230:460–8. [16] Lo H-S, Reeves RE. Purification and properties of NADPH:flavin oxidoreductase from Entamoeba histolytica. Mol Biochem Parasitol 1980;2:23–30. [17] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248 – 54. [18] Shim H, Fairlamb AH. Levels of polyamines, glutathione and glutathione-spermidine conjugates during growth of the insect trypanosomatid Crithidia fasciculata. J Gen Microbiol 1988;134:807–17.

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[19] Fairlamb AH, Henderson GB, Bacchi CJ, Cerami A. In vivo effects of difluoromethylornithine on trypanothione and polyamine levels in bloodstream forms of Trypanosoma brucei. Mol Biochem Parasitol 1987;24:185 – 91. [20] Borges A, Cunningham ML, Tovar J, Fairlamb AH. Site-directed mutagenesis of the redox-active cysteines of Trypanosoma cruzi trypanothione reductase. Eur J Biochem 1995;228:745 – 52. [21] Hunter KJ, Le Quesne SA, Fairlamb AH. Identification and biosynthesis of N 1,N 9-bis(glutathionyl)aminopropylcadaverine (homotrypanothione) in Trypanosoma cruzi. Eur J Biochem 1994;226:1019 – 27. [22] Newton GL, Fahey RC. Determination of biothiols by bromobimane labeling and high-performance liquid chromatography. Methods Enzymol 1995;251:148 – 66. [23] Newton GL, Fahey RC. Purification of thiols from biological samples. Methods Enzymol 1987;143:96 – 101. [24] Fahey RC, Sundquist AR. Evolution of glutathione metabolism. Adv Enzymol Relat Areas Mol Biol 1991;64:1 – 53. [25] Margulis L, Chapman MJ. Endosymbioses: cyclical and permanent in evolution. Trends Microbiol 1998;6:342 – 5. [26] Margulis L. Archaeal-eubacterial mergers in the origin of Eukarya: phylogenetic classification of life. Proc Natl Acad Sci USA 1996;93:1071 – 6. [27] Bakker Grunwald T, Wostmann C. Entamoeba histolytica as a model for the primitive eukaryotic cell. Parasitol Today 1993;9:27 – 31. [28] Clark CG, Roger AJ. Direct evidence for secondary loss of mitochondria in Entamoeba histolytica. Proc Natl Acad Sci USA 1995;92:6518 – 21. [29] Roger AJ, Svard SG, Tovar J, Clark CG, Smith MW, Gillin FD, Sogin ML. A mitochondrial-like chaperonin 60 gene in Giardia lamblia: evidence that diplomonads once harbored an endosymbiont related to the progenitor of mitochondria. Proc Natl Acad Sci USA 1998;95:229 – 34. [30] Gray MW, Burger G, Lang BF. Mitochondrial evolution. Science 1999;283:1476– 81. [31] Bruchhaus I, Richter S, Tannich E. Recombinant expression and biochemical characterization of an NADPH:flavin oxidoreductase from Entamoeba histolytica. Biochem J 1998;330:1217– 21. [32] Bruchhaus I, Richter S, Tannich E. Removal of hydrogen peroxide by the 29 kDa protein of Entamoeba histolytica. Biochem J 1997;326:785 – 9. [33] Gutteridge WE, Coombs GH. Catabolism and the generation of energy. I. Stages parasitic in vertebrates. In: Gutteridge WE, Coombs GH, editors. Biochemistry of Parasitic Protozoa. London: Macmillan, 1977:32 – 58. [34] Zilberstein D, Dwyer DM. Glucose transport in Leishmania dono6ani promastigotes. Mol Biochem Parasitol 1984;12:327 – 36.