13 Anti-oxidant enzymes in Cryptosporidium parvum oocysts
E. ENTRALA", C. MASCARO" and J. BARRETT#* "Departmento Parasitologia, Facultad de Ciencias, Campus Fuentenueva, E-18071 Granada, Spain # Institute of Biological Sciences, University of Wales, Aberystwyth, Dyfed SY23 3DA
(Received 15 April 1996; revised 1 July 1996; accepted 2 July 1996)
Oocysts of Cryptosporidium parvum showed relatively low levels of SOD activity. The SOD which had a pI of 4n8 and an approximate molecular weight of 35 kDa appeared to be iron dependent. Catalase, glutathione transferase, glutathione reductase and glutathione peroxidase activity could not be detected, nor could trypanothione reductase. No NADH or NADPH oxidase activity could be detected, nor could peroxidase activity be demonstrated using o-dianisidine, guaiacol, NADPH or NADH as co-substrates. However, an NADPH-dependent H#O# scavenging system was detected in the insoluble fraction.
Key words: Cryptosporidium parvum, anti-oxidant enzymes, SOD.
diluted with saline solution (0 9% NaCl, w\v), the n gross material was removed by filtration and the The role of cell-mediated immunity in host re- samples washed by centrifugation (3500 g for sistance to intracellular pathogens is well established 15 min) and resuspended in saline solution until a (Nathan et al. 1979; Meshnick & Eaton, 1981; low viscosity suspension was obtained. The C. Murray, 1981, 1983; Britten & Hughes, 1986; parvum oocysts were purified using a caesium Sibley, Lawson & Weidner, 1986). The anti- chloride gradient (Kilani & Sekla, 1987) and stored microbial activity of macrophages and mast cells is in saline at 4 mC. closely tied to oxygen radical production, triggered during phagocytosis or activation. Hydrogen per- oxide, superoxide radicals and hydroxyl radicals are Preparation of cell-free homogenate also generated as products of normal cellular metab- A number of different techniques were tried and the olism. Cells are protected from the damaging effects following gave the highest protein recovery. Purified of reactive oxygen intermediates by scavengers and C. parvum oocysts were excysted according to the specific enzymes such as catalase, superoxide dis- method described by Robertson, Campbell & Smith mutase, glutathione peroxidase and glutathione S- (1993). Briefly, oocysts were suspended in Hanks transferase. balanced salt solution (HBSS) containing 1% (w\v) Little is known about the ability of parasitic trypsin and adjusted to pH 2n75 with 1 HCl. After protozoans to cope with reactive oxygen species 1 h at 37 mC the oocysts were centrifuged (2000 g for generated during inflammation or when phago- 15 min at 4 mC), resuspended in bile solution (1% cytosed by macrophages. In this paper we report (w\v) bovine bile and 0n06% (w\v) NaHCO$ in on the anti-oxidant enzymes in Cryptosporidium HBSS) and incubated at 37 mC for 30 min. Oocyst parvum. This is a protozoan which parasitizes the excystation and sporozoite motility was verified epithelial tissues of a wide range of vertebrates, microscopically, before concentration by centri- including man, where it can cause a chronic infection fugation (1500 g for 15 min at 4 mC). The pellet was in immuno-compromised individuals. washed twice with HBBS then resuspended in homogenization buffer (0n25 sucrose, 2 m EDTA, 0n1% (v\v) Triton X-100 in 10 m phos- phate buffer, pH 7n2) and sonicated for 5 min with an Parasite material MSE 150 Watt ultrasonic disintegrator Mk2 at 70% Faecal samples were collected from naturally in- power. After centrifugation at 10000 g for 10 min at fected newborn Holstein calves. Samples were 4 mC previously washed Carborundum powder (extra fine, 300 grit, Fisons UK) was added to the precipitate and any intact oocysts were disrupted by * Corresponding author: Institute of Biological Sciences, UWA, Aberystwyth, Dyfed SY23 3DA. Tel: 01970- grinding in a mortar. The viscous paste was diluted 622315. Fax: 01970-622350. E-mail: jzb!aber.ac.uk. with buffer, the Carborundum powder was allowed
Parasitology (1997), 114, 13–17 Copyright # 1997 Cambridge University Press E. Entrala, C. Mascaro and J. Barrett 14 to sediment and the sample was again centrifuged at SOD activity is defined as the amount of enzyme 10000 g for 10 min at 4 mC and the soluble fraction required to inhibit the rate of NAD(P)H oxidation of added to the previous supernatant to give a whole the control by 50%. homogenate. The homogenate was concentrated and SOD activity was also demonstrated using an on low molecular weight compounds (which interfered gel assay. The proteins were separated on 8–25% with the superoxide dismutase assay) removed by native polyacrylamide gradient gels and on pH 3 ultrafiltration in Centricon-10 microconcentrators isoelectric focusing gels using a Pharmacia Phast- (cut off 10 kDa, Amicon). Fractions were stored in system. SOD activity staining was carried out with liquid nitrogen until used. the same reagents as the NBT assay. Gels were Protein was determined with Coomasie Brilliant soaked in 10 ml of the stock solution plus 0n1mlof Blue G250 (Sedmak & Grossberg, 1977) using the 0n1mriboflavin solution and illuminated with bovine serum albumin as the standard. UV light until enzyme activity appeared as a colourless band on a blue background. To dis- tinguish between Cu–Zn and Mn or Fe dependent Enzyme assays SOD, activity was measured in the presence of 1 m All assays were started by the addition of enzyme potassium cyanide (2 m for the on gel assay) and after treatment with 5 m H O for 5 min at 37 C and conducted at 25 mC unless otherwise stated, # # m concentration of reagents are final concentrations. (5 m H#O#,1mEDTA for 45 min at 37 mC for the on gel assay). Bovine erythrocyte superoxide dismutase together with the Mn-SOD and Fe-SOD Catalase (EC 1.11.1.6). The reaction mixture con- from E. coli (Sigma) were used as positive controls. tained (in 1 ml): 10 m H#O#,50mpotassium Molecular weight and pI gel markers were visualized phosphate buffer, pH 7n2. The decrease in OD was by silver staining. followed at 240 nm for 1 min.
Glutathione reductase (EC 1.6.4.2). The reaction Superoxide dismutase (EC 1.15.1.1). SOD was de- mixture contained (in 1 ml): 150 m potasium termine by the method of Beyer & Fridovitch (1987). phosphate buffer, pH 7n2, 1 m EDTA, 0n14 m In this assay superoxide anions generated by the NADPH, 1 m glutathione (oxidized). The decrease photoactivation of riboflavin reduces nitroblue- in OD was followed at 340 nm. tetrazolium (NBT) to an insoluble purple formazan. One ml of stock solution (containing 27 ml of 50 m Glutathione peroxidase (EC 1.11.1.9). The assay was potassium phosphate buffer, pH 7n8, 1n5mlof0n2 based on that described by Flohe & Gunzler (1984). -methionine, 1n0mlof1n6mNBT and 0n75 ml of 1% (v\v) Triton X-100) and serial dilutions of The reaction mixture contained (in 1 ml): 50 m homogenate (20–100 µl) were placed in a series of potassium phosphate buffer, pH 7n0, 0n5mEDTA, 0 24 units glutathione reductase (Sigma), 5 m cuvettes together with 10 µlof0n1m riboflavin n solution to initiate the reaction. After mixing, the glutathione (reduced). The mixture was allowed to cuvettes were illuminated for 15 min under UV light equilibrate at 37 mC for 10 min, then 0n1mlof1n5m (controls without homogenate were run in parallel). NADPH in 100 m NaHCO$ was added and the The OD at 560 nm was measured before and after peroxide-independent oxidation of NADPH fol- incubation. The rate (control divided by inhibited) lowed at 340 nm for 3 min. The reaction was started was plotted against protein concentration and by the addition of 0n1 ml of pre-warmed 1n5m units\mg protein determined (1 unit of SOD is H#O#(for selenium-dependent activity) or 0n1mlof defined as the amount required to cause 50% 12 m t-butyl hydroperoxide (for non-selenium inhibition in the reduction of NTB). dependent activity). SOD activity was also determined by the method of Paoleti & Mocali (1990). This is considerably Glutathione S-transferase (EC 2.5.1.18). The assay more sensitive than the NBT assay and involves the was essentially as described by Habig, Pabst & oxidation of NAD(P)H by superoxide anions gen- Jakoby (1974) using 1-chloro-2,4-dinitrobenzene erated chemically from molecular oxygen in the (CDNB) or 1,2-epoxy-3-(p-nitrophenoxy)propane presence of manganous ions. The reaction mixture (ENP) as substrate. The reaction mixture contained contained in 1 ml: 80 m triethanolamine- (in 1 ml): 100 m potassium phosphate buffer, 1 m diethanolamine buffer, pH 7n4, 0n4m NAD(P)H, glutathione (reduced) for the CDNB assay, or 5 m 2n8mEDTA, 1n4mMnCl# and 0n1 ml of sample for the ENP assay. The reaction was started after (or buffer control). The cuvettes were mixed and the 2–3 min pre-incubation by the addition of 1 m OD recorded at 340 nm for 5 min to obtain a stable CDNB or 5 m ENP. The formation of the baseline. The reaction was then started by the glutathione conjugate was followed at 340 nm for −" −" addition of 0n1mlof10m2-mercaptoethanol and CDNB (ε l 9n6m cm ) and at 360 nm for ENP −" −" the decrease in OD at 340 nm followed. One unit of (ε l 0n5m cm ). Anti-oxidants in C. parvum 15
Fig. 1. Homogenates of Cryptosporidium parvum were separated on isoelectrofocusing (pH 3–9) gels and SOD visualized by an on-gel assay. (A) Without inhibitors; (B) with 2 m KCN; (C) with 5 m H#O#,1mEDTA for 45 min at 37 mC. Lanes 1 and 2, C. parvum; Lane 3, Cu\Zn SOD (bovine); Lane 4, Mn-SOD (E. coli); Lane 5, Fe- SOD (E. coli).
Trypanothione reductase. The reaction mixture con- components. Activity was not inhibited by 1 m tained (in 1 ml): 100 m HEPES buffer, pH 7n2, KCN, but was partially inhibited by pre-incubation 0n1m EDTA, 0n5m oxidized trypanothione, with 0n5m hydrogen peroxide (23% and 33% 0n25 m NAD(P)H. The decrease in OD was after pre-incubation for 5 min and 30 min respect- followed at 340 nm. ively) suggesting an iron-dependent SOD. From native PAGE, the molecular weight of the Peroxidase activity (EC 1.11.1.7). This was assayed C. parvum SOD was estimated at 35 kDa and it spectrophotometrically as described by Paul & appeared as a single band on isoelectric focusing gels Barrett (1980) using guaiacol as the substrate. The with a pI of 4n8. The activity stain was not affected by on-gel assay described by Brown, Upcroft & Upcroft pre-incubation of the gel with 2 m KCN, but was (1995) was also used to assay for peroxidase using o- inhibited when the gel was pre-incubated in phos- dianisidine as the substrate and for NADH and phate buffer containing 5 m H#O#,0n1mEDTA NADPH peroxidase and NADH and NADPH for 45 min at 37 mC (Fig. 1). These results are again oxidase activities. consistent with the presence of an iron-containing SOD, similar to that described from other parasitic protozoans (Meshnick & Eaton, 1981; Le Trant et al. 1982; Kitchner et al. 1984; Sibley et al. 1986; The oocysts of C. parvum showed only low levels of Becuwe et al. 1992). SOD activity in C. parvum SOD activity. Using the NBT assay a value of oocysts was first described by Ogunkolade et al. 5n7p1n4 units\mg protein (n l 3) was found in (1993) as a rapidly fading band on starch gels. In the dialysed samples of the soluble fraction. This activity present assay, the band was stable for at least 2 was destroyed by heating to 100 mC for 5 min. months. Undialysed samples showed an activity of 22n6p2 There have been few studies on SOD from units\mg protein but 98% of the activity was coccidia. Toxoplasma gondii has an Fe-SOD (Sibley retained after boiling for 30 min. The undialysed et al. 1986), but the activity is higher than in C. insoluble fraction also showed apparent SOD ac- parvum.InEimeria tenella Fe-SOD only represents tivity (11n4p6n4 units\mg protein), but again the 30–40% of the total SOD activity in unsporulated activity was not destroyed by heating to 100 mC for oocysts, copper-zinc and manganese-dependent 30 min. The apparent heat-stable SOD activity is enzymes also being present (Michalski & Prowse, due to the presence of free radical scavenging agents 1991). In Eimeria SOD activity decreases during such as mannitol or alpha tocophoral. Using the sporulation (from 380 to 9n8 units\mg) and only the NAD(P)H oxidation assay, the mean SOD activity manganese-SOD is present in fully sporulated in the dialysed soluble fraction was 53n2p4n2 oocysts and sporozoites. units\mg protein. The higher activity with the No catalase activity could be detected either NAD(P)H assay probably reflects the fact that this spectrophotometrically or by the on-gel assay (lower assay is less prone to interference by other cellular limit of detection 0n5 nmoles\min\mg protein). A E. Entrala, C. Mascaro and J. Barrett 16 decrease in OD at 240 nm in the presence of , . ., , . . , . (1995). Free hydrogen peroxide caused by C. parvum extracts was radical detoxification in Giardia duodenalis. Molecular found not to be destroyed by boiling and did not and Biochemical Parasitology 72, 47–56. follow first-order kinetics. This suggests that the , . . , . . (1993). Thermal decrease in OD was not due to catalase, but to the regulation of active oxygen-scavenging enzymes in Crithidia luciliae thermophila. Journal of Parasitology direct chemical oxidation, by hydrogen peroxide, of 79, 809–814, an endogenous substrate in the parasite extract. This , ., -, ., , . , . ‘non-specific’ catalase activity appeared to be asso- (1995). Influence of hydrogen peroxide on acid-fast ciated with the insoluble fraction and it is interesting staining of Cryptosporidium parvum oocysts. to note that Entrala et al. (1995) found that hydrogen International Journal for Parasitology 25, 1473–1477. peroxide altered the staining characteristics of C. , . ., , ., , ., , . . parvum oocysts. , . . (1988). Oxidant defense enzymes of No NADPH or NADH oxidase activity could be Plasmodium falciparum. Molecular and Biochemical detected in extracts of C. parvum oocysts, nor could Parasitology 30, 77–82. NADPH or NADH peroxidase activity or organic , . , . . (1984). Assays of glutathione peroxidase activity be demonstrated (lower limits of peroxidase. Methods in Enzymology 105, 114–121. , . ., , . . , . . (1974). detection 0n1–0n5 nmoles\min\mg protein). Glutathione S-transferases. The first enzymatic step Glutathione transferase, glutathione reductase and in mercapturic acid formation. Journal of Biological glutathione peroxidase activity were not detected in Chemistry 249, 7130–7139. C. parvum oocysts nor could trypanothione reductase , . . , . (1987). Purification of activity be detected using either NADPH or NADH Cryptosporidium oocysts and sporozoites by cesium (lower limit of detection 0n5 nmoles\min\mg pro- chloride and Percoll gradients. American Journal of tein).However, NADPH-dependent H#O# consump- Tropical Medicine and Hygiene 36, 505–508. tion was detected in the insoluble fraction , . ., , . ., , . . (34n6p11n6 µmol\min\mg, n l 4). Similar levels of , . . (1984). An iron-containing superoxide activity have been reported in isolated late stage dismutase in Tritrichomonas foetus. Molecular Plasmodium falciparum (Fairfield et al. 1988) as well Biochemistry and Parasitology 12, 95–99. as in cultured Crithidia luciliae luciliae and Crithidia , ., , . ., , ., , luciliae thermophila (Emtage & Bremner, 1993). In . . , . (1982). Iron-containing superoxide dismutase from Crithidia fasciculata. Purification, these trypanosomatides NADPH-dependent H O # # characterisation, and similarity to leishmanial and scavenging systems seem to be more important than trypanosomal enzymes. Journal of Biological Chemistry catalase, at least in response to heat stress. 258, 125–130. C. parvum lacks mitochondria. The absence of , . . , . . (1981). Leishmanial catalase and peroxidase activity and the apparent superoxide dismutase: a possible target for absence of glutathione and trypanothione dependent chemotherapy. Biochemical and Biophysical Research enzyme systems would suggest that there is no Communications 102, 970–976. classical respiratory transport chain in this parasite. , . . , . . (1991). Superoxide Oxidative protection must be being provided by dismutase in Eimeria tenella. Molecular and alternative mechanisms such as high concentrations Biochemical Parasitology 47, 189–196. of free-radical scavengers such as mannitol or , . . (1981). Susceptibility of Leishmania to alternative thiols. oxygen intermediates and killing by normal macrophages. Journal of Experimental Medicine 153, 1302–1315. This work was supported by a Human Capital and , . . (1983). Macrophage oxygen-dependent Mobility Grant from the EC. killing of intracellular parasites: Toxoplasma and Leishmania. Advances in Experimental Biology and Medicine 162, 127–143. , ., , ., , ., , . , ., , ., , ., , ., , . (1979). Activation of macrophages in vivo , . , . (1992). Endogenous superoxide and in vitro. 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