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Volume 221, number 2, 427-431 FEB 05098 September 1987

Trypanosomatid hydrogen metabolism

P.G. Penketh, W.P.K. Kennedy*, C.L. Patton and A.C. Sartorelli

MacArthur Center for Parasitology and Tropical Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06510, USA and * Wolfson Molecular Biology Unit, London School of Hygiene and Tropical Medicine, Keppel Street, London WCLE 7HT, England

Received 7 July 1987

The rate of whole cell HZ02 metabolism in several salivarian and stercorarian trypanosomes and Leishrnania species was measured. These cells metabolized Hz02 at rates between 2.3 and 48.2 nmol/lO* cells per min depending upon the species employed. Hz02 metabolism was largely insensitive to NaN3, implying that typi- cal and peroxidase haemoproteins are not important in Hz02 metabolism. The metabolism of H202, however, was almost completely inhibited by N-ethylmaleimide. In representative species, Hz02 metabolism was shown to occur through a trypanothione-dependent mechanism.

Hydrogen ; Trypanothione; Macrophage; (Trypanosoma, )

1. INTRODUCTION been reported that L. donovani possesses a surface-membrane acid phosphatase which reduces Many trypanosomatids have been reported to the magnitude of the oxidative burst of lack or to be extremely deficient in enzyme systems neutrophils, inferring a pathophysiological role for necessary for the removal of Hz02 (i.e. catalase, this enzyme [5]. However, defense mechanisms peroxidase) [l-5]. Defense against endogenously generated Hz02 and the mechanisms against H202, however, appear to be possibly diminished quantity of Hz02 produced a ubiquitous requirement of most aerobic cells [6]. during the oxidative burst would still be required. Leishmania and Trypanosoma cruzi Recently, T. brucei has been shown to contain a .trypomastigotes must also withstand the oxidative novel enzymic HzOz-metabolizing system. This killing mechanism of phagocytic macrophages and system utilizes NADPH for the reduction of Hz02 other host cells [7]. This suggests the presence of and has an absolute requirement for the dithiol co- mechanisms other than catalase and glutathione factor trypanothione [N’,M-bis(L-y-glutamyl-L- peroxidase activities to fulfill this vital role. It has hemicysteinylglycyl)] [9, lo]. Similar ac- tivity was measured when organic hydroperoxides Correspondence address: P.G. Penketh, MacArthur were used as substrates, implying that an addi- Center for Parasitology and Tropical Medicine, Yale tional role exists for this activity in the reduction of University School of Medicine, 333 Cedar Street, New lipid hydroperoxides [lo]. The precise details of Haven, CT 06510, USA the reaction mechanisms involved are not yet clear. A system analogous to the host glutathione reduc- Abbreviations: BSA, bovine serum albumin; NEM, N- ethylmaleimide; GSSWGSH, oxidized and reduced tase/glutathione peroxidase system utilizing glutathione, respectively; NADH, reduced nicotinamide trypanothione was anticipated. adenine dinucleotide; NADP+/NADPH, oxidized and Trypanothione reductase has been purified to reduced nicotinamide adenine dinucleotide phosphate, homogeneity from several trypanosomatides respectively; TWT(SHl2, oxidized and reduced [ 11,121. Trypanothione peroxidase activity has trypanothione, respectively been inferred in crude extracts or crude extracts

Published by Elsevier Science Publishers B. V. (Biomedical Division) 00145793/87/$3.50 0 1987 Federation of European Biochemical Societies 427 Volume 221, number 2 FEBS LETTERS September 1987 supplemented with additional trypanothione amastigotes were obtained and purified from in- reductase by a presumptive coupled enzymic assay fected Syrian hamster spleens as described by [9,10,13]; however, the expected trypanothione Channon et al. [19]. L. braziliensis and L. mex- peroxidase activity has so far eluted detection by icana amazonensis promastigotes were cultured in direct assay in both T. brucei and T. cruzi Schneider’s medium containing 2 mM glutamine, [9,10,14]. The following reaction scheme has been 10% fetal calf serum, 100 U/ml of penicillin and proposed for the metabolism of hydroperoxides 100 pg/ml of streptomycin at 25°C. L. braziliensis [13]. amastigotes were grown in culture as described by Pan [20]. L. tarantolae was cultured in LT medium ROOH ROH i- Hz0 at 27°C. All cells were washed in incubation buffer (5 mM KCl, 80 mM NaCl, 2 mM MgC12, 16.2 mM Na2HP04, 3.8 mM NaHzP04, 50 mM D-glucose, 0.27 mM phenol red and 1.5 mg/ml of BSA, ad- $r y panot hione per oxid justed to pH 7.4) and stored on ice prior to use. Hz02 metabolism of cell suspensions was measured as described in the legend to table 1. NADPH dependent Hz02 consumption was measured as follows. Trypanosomes were washed in glucose-free incubation buffer, incubated for 10 min at room temperature, pelleted by cen- trifugation (3000 x g for 10 min) and osmotically lysed by suspension in distilled water at a density equivalent to lo9 cells/ml. This suspension was NADP+ NADPH + H+ centrifuged at 20000 x g for 10 min and 1 vol. of 1 M Tris-HCl (pH 8.0) containing 10 mM EDTA Here, we have measured the ability of 12 buffer was added to 9 vols of the supernatant. trypanosomatids to metabolize H202. NADPH and NADP+ were added to give final concentrations of 100 PM (the inclusion of 100 FM 2. MATERIALS AND METHODS NADP+ corrects for any residual NADP+ reduc- ing capacity). Hz02 was then added to the sample Enzymes and co-factors were obtained from cuvette at concentrations between 2.5 and 20,uM Sigma. Synthetic trypanothione was a kind gift and the change in absorbance at 340 nm was from Dr A. Fairlamb. All other chemicals were of followed at 37°C against an equivalent sample AR grade. Incubation buffer was composed as in without H202. This procedure was further [lOI. modified in the case of T. lewisi because of the Bloodstream trypomastigotes of T. brucei high NADP+ reducing capacity of these lysates. rhodesiense (YTat 1. I), T. equiperdum, T. evansi These cells were lysed at a level of 5 x 10’ cells/ml and T. vivax were prepared and purified as and 250 PM NADP+ was used instead of 100 PM described by Lanham [15]. T. lewisi and T. con- NADPH and 1OOpM NADP+. Hz02 was then golense were prepared in a similar manner, except added after the mixture had been incubated for that lethally irradiated rats were used as hosts. 5 min. L. mexicana amazonensis promastigotes Procyclic forms of T. brucei rhodesiense were were treated in a manner analogous to that of T. cultured in Cunningham’s medium supplemented lewisi except that these cells, due to their resistance with 10% fetal calf serum at 27°C. T. cruzi Y to osmotic lysis, were lysed at a level of lo9 strain and X 10 strain epimastigotes were cultured cells/ml by nitrogen cavitation in distilled water. as described by Widmer [ 161. T. cruzi In all of these preparations, the intrinsic trypomastigotes (Y strain) were prepared ac- trypanothione reductase activity greatly exceeded cording to Piras et al. [17]. L. donovani promas- that of the peroxidase; for example, a 50-fold dif- tigotes were cultured and isolated as described by ference in activity has been reported in T. brucei Neal [ 181 for L. tropica major. L. donovani [13]. The HzOz-mediated oxidation of NADPH

428 Volume 221, number 2 FEBS LETTERS September 1987

Table 1 were prepared using an Amicon PM-10 filter The initial rate of Hz02 metabolism by various (nominal molecular mass cut off at 10 kDa). The trypanosome and Leishmaniu species high molecular mass fraction was washed with 2-times the initial lysate volume of distilled Hz0 Organism nmol Hz02 con- and suspended in a small volume of H20. Catalase sumed per 10’ cells activity was measured using an 02 electrode as in during the 1st min [91. of exposure to 20rM Hz02 (*SD) 3. RESULTS AND DISCUSSION

Salivarian trypanosomes All of the cell lines studied were able to T. brucei rhodesiense metabolize Hz02 (table 1). The high consutiption trypomastigotes 3.5 k 0.6 (5) of Hz02 observed in T. cruzi trypomastigotes com- T. brucei rhodesiense pared to epimastigote forms is consistent with their procyclic 6.4 f 1.2 (5) greater resistance to the phagocytic killing T. equiperdum trypomastigotes mechanism and to oxidant stress in the form of Lump 1559 3.7 -t 0.5 (4) Hz02 [21]. The various forms of L. donovani, L. T. evansi SN trypomastigotes mexicana, L. braziliensis, T. cruzi and T. lewisi Lump 1559 3.5 Ik 0.3 (5) studied showed a progressive decrease in the rate T. evansi SAK trypomastigotes 4.2 f 0.3 (5) T. vivax trypomastigotes 3.5 f 0.2 (3) of Hz02 consumption at relatively low (< 10 PM) T. congolense trypomastigotes concentrations of H202, whereas the salivarian TREU 1290 3.6 + 0.4 (3) trypanosomes gave more linear kinetics (not shown). Hz02 metabolism was largely insensitive Storcorarian trypanosomes to 1 mM NaN3, except for L. donovani T. lewisi trypomastigotes 19.6 + 3.0 (7) amastigotes, which implied that typical T. cruzi trypomastigotes Y strain 48.2 + 1.9 (3) catalase/peroxidase haemoproteins are not impor- 18.3 k 1.5 (3) T. cruzi epimastigotes Y strain tant in the metabolism of H202. Hz02 metabolism, T. cruzi epimastigotes however, was inhibited 85-100% by pretreatment X 10 strain 4.2 f 0.2 (3) with NEM (table 2). L. donovani amastigotes Leishmania behaved differently, in that a 40% inhibition of the L. donovani promastigotes 4.3 f 0.2 (3) initial rate of Hz02 metabolism was produced by L. donovani amastigotes 2.3 k 0.2 (3) 1 mM NaN3, suggesting the presence of a catalase- L. brasiliensis promastigotes 8.3 f 0.3 (3) type activity. An NaN3-sensitive catalase activity L. brasiliensis amastigotes 2.3 + 0.4 (3) was confirmed in L. donovani amastigote osmotic L. mexicana amazonensis lysates using an 02 electrode. This catalase showed promastigotes 5.6 f 0.7 (3) an identical electrophoretic mobility to that of 3.3 + 1.1 (4) L. turentolae (culture forms) Syrian hamster spleen catalase when localized on Freshly washed cells were suspended at concentrations starch gels (Kennedy, W.P.K., unpublished), sug- of lo’-10’ cells/ml, depending upon the species, to gesting contamination by host enzyme. Catalase is obtain conveniently measurable rates in incubation a very ‘sticky’ enzyme and tends to become attach- buffer at 37°C. Hz02 was added to give a final ed to the outside of organelles during tissue concentration of 20pM and 1.2-ml aliquots were homogenization. This has led to reports that removed at I-min intervals and assayed for residual mitochondria and chloroplasts contained catalase Hz02 as described [9]. The values in parentheses indicate activity. Careful experimentation, however, con- the number of samples assayed firmed the absence of catalase inside these organelles [22,23]. A similar phenomenon may be occurring during the preparation of amastigotes. was independent of the NADPH concentration In support of this premise, no catalase activity was within the tested range (i.e. 50-2OOpM). found in in vitro cultured amastigotes and pro- Ultrafiltrates and high molecular mass fractions mastigotes of L. braziliensis.

429 Volume 221, number 2 FEBS LETTERS September 1987

Table 2 concentrations that are lower than those of the ex- The effects of the presence of NaN3 and pretreatment tracellular environment. with NEM on the initial rate of Hz02 metabolism by Cells lysed at low concentrations showed a various trypanosome and Leishmania species disproportionate decrease in activity in this assay, due in part to the presence of subsaturating con- Organism Relative ac- Relative ac- centrations of trypanothione. Under the condi- tivity in the tivity after tions employed, approx. 30% of the intact cellular presence of NEM pre- activity was retained. No activity was observed NaNs treatment when NADH was substituted for NADPH. T. brucei rhodesiense trypomasti- Large reserves of a compound(s) supporting gotes 1.03 0.02 NADP+ reduction was observed in L. mexicana T. equiperdum trypomasti- promastigotes and in T. lewisi. These reserves gotes Lump 1559 0.97 0.00 could be important in sustaining the defense T. evansi SN trypomasti- against prolonged oxidant stress by H202. Very gotes 0.97 0.00 large reserves have also been measured in lysates T. evansi SAX trypo- prepared from L. donovani promastigotes and 0.03 mastigotes 1.00 from T. cruzi epimastigotes, sufficient to reduce T. vivax trypomastigotes 0.86 0.03 about 30 nmol NADP+/lO* cells [lo]. T. cruzi epimastigotes X 10 strain 1.05 0.15 High molecular mass fractions and ultrafiltrates T. fewisi trypomastigotes 0.89 0.00 prepared from blood-stream trypomastigote forms L. donovani promastigotes 1.02 0.07 of T. equiperdum; T. lewisi and L. mexicana L. donovani amastigotes 0.61 0.39 amazonensis promastigotes individually showed L. braziliensis promastigotes 0.94 0.09 no HzOz-dependent NADPH oxidation. However, L. braziliensis amastigotes 0.90 0.00 when the ultrafiltrates and high molecular mass fractions were recombined, activity was restored in The initial rate of Hz02 metabolism was measured as described in the legend to table 1 in the presence of a manner approximately proportional to the con- 1 mM NaNs and also after pretreatment of cells with centration of the high molecular mass fraction. 0.2 mM NEM at 22°C for 50 min in incubation buffer. Addition of GSH (100 PM) and GSSG reductase Activity is expressed relative to that of untreated (0.1 U/ml) to the high molecular mass fraction did controls, which were equated to 1.0 not restore activity, indicating that a GSH perox- idase system was not operative. The addition of concentrated T. brucei ultrafiltrate or synthetic NADPH-dependent Hz02 consumption was trypanothione restored activity in a concentration measured in lysates prepared from blood-stream dependent saturable manner, with l/2 V,,,,, being forms of the following species: T. brucei rhode- achieved between 1 and 10pM trypanothione, siense, T. evansi, T. equiperdum, T. vivax, T. depending upon the species employed. lewisi and in promastigote forms of L. mexicana Ultrafiltrates from the above species restored ac- amazonensis. Addition of Hz02 in all cases tivity in T. brucei high molecular mass fractions. resulted in a stoichiometric and linear (down to These findings indicate that the novel process about 1 PM H202) oxidation of NADPH. The responsible for Hz02 metabolism in T. brucei is stoichiometric consumption of NADPH implies common to the above species. A low glutathione that NADPH-independent Hz02 consumption is peroxidase activity has been reported in L. unimportant in these lysates. The linear rate of donovani by Murray [24]; this contrasts with the NADPH oxidation that was observed suggests the findings of Channon and Blackwell [5] who in- presence of an enzymic process with a low I&, dicated that this enzyme was absent. However, the value (< 1 PM) for Hz02 and supports the idea that assay used by Murray contained some endogenous the rate-limiting diffusion of Hz02 into cells is the trypanothione which may have accounted for the cause of the non-linear kinetics seen with intact small activity that was observed. The importance cells at low Hz02 concentrations. This and probable general occurrence of this novel phenomenon would result in intracellular Hz02 trypanothione-dependent process in many if not all

430 Volume 221, number 2 FEBS LETTERS September 1987 trypanosomatids make it an ideal target for a Basfora, R.E., Waggoner, A.S., Ernst, L.A. and directed chemotherapeutic attack. Pope, M. (1985) Exp. Parasitol. 60, 333-341. 191 Penketh, P.G. and Klein, R.A. (1986) Mol. Biochem. Parasitol. 20, 111-121. ACKNOWLEDGEMENTS WI Penketh, P.G. (1986) in: Nitrofurazone and Metabolism in Trypanosoma P.G.P., C.L.P. and A.C.S. acknowledge the brucei, PhD Thesis, Cambridge University. support of USPHS grant AI 15742 and a grant 1111 Krauth-Siegel, R.L., Enders, B., Henderson, G.B., from the MacArthur Foundation. W.P.K.K. was Fairlamb, A.H. and Schirmer, R.H. (1987) Eur. J. supported during these studies by the Wolfson Biochem. 164, 123-128. Foundation. WI Shames, S.L., Fairlamb, A.H., Cerami, A. and Walsh, T. (1986) Biochemistry 25, 3519-3526. u31 Henderson, G.B., Fairlamb, A.H. and Cerami, A. REFERENCES (1987) Mol. Biochem. Parasitol. 24, 39-45. 1141 Schirmer, R.H., Schollhammer, T., Eisenbrand, [II Docampo, R. and Moreno, S.N.J. (1984) in: Free G. and Krauth-Siegel, R.L. (1987) Free ,Radical Radicals in Biology, pp.243-288, Academic Press, Res. Commun. 3, 3-12. New York. u51 Lanham, S.M. (1968) Nature 218, 1273-1274. 121 Boveris, A., Seis, H., Martino, E.E., Docampo, [I61 Widmer, G. (1986) in: A Biochemical and R., Turrens, J.F. and Stoppani, H.O.M. (1980) J. Epidemiological Study of Enzyme Polymorphism Biochem. 188, 643-648. in Trypanosoma cruzi, PhD Thesis, London School 131 Clark, LA., Hunt, N.H. and Cowden, W.B. (1986) of Hygiene and Tropical Medicine. Adv. Parasitol. 25, l-28. I171 Piras, M.M., Piras, R. and Henriques, D. (1982) 141 Channon, J.Y. and Blackwell, J.M. (1985) Mol. Biochem. Parasitol. 6, 67-81. Parasitology 91, 197-200. 1181 Neal, R.H. (1984) Exp. Parasitol. 57, 269-273. 151 Channon, J.Y. and Blackwell, J.M. (1985) [I91 Channon, J.Y., Roberts, M.V. and Blackwell, Parasitology 91, 207-217. J.M. (1984) Immunology 52, 345-355. 161 Halliwell, B. (1981) in: Free Radicals, Oxygen PO1 Pan, A.A. (1984) Exp. Parasitol. 58, 72-80. Toxicity and Aging, pp. l-62, Elsevier, WI Tanaka, Y., Tanowitz, H. and Bloom, B.R. (1983) Amsterdam, New York. Infect. Immun. 41, 1322-1331. [71 Nguyen, L.T., Meshnick, S.R., Kitchener, K., WI Day, D.A., Arron, G.P. and Laties, G.G. (1979) J. Eaton, J.W. and Cerami, A. (1983) J. Biol. Chem. Exp. Biol. 30, 539-549. 258, 125-130. v31 Allen, J.F. (1977) FEBS Lett. 84, 221-224. PI Remaley, A.T., Glew, R.H., Huhns, D.B., v41 Murray, H.W. (1982) J. Immunol. 129, 351-357.

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