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 4±8 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 ± 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 2±75 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 0±06% (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 (0±25 sucrose, 2 m EDTA, 0±1% (v}v) Triton X-100 in 10 m phos- phate buffer, pH 7±2) 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 0±1mlof Blue G250 (Sedmak & Grossberg, 1977) using the 0±1mriboflavin 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 7±2. 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 7±2, 1 m EDTA, 0±14 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 7±8, 1±5mlof0±2 based on that described by Flohe & Gunzler (1984). -methionine, 1±0mlof1±6mNBT and 0±75 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 7±0, 0±5mEDTA, 0 24 units glutathione reductase (Sigma), 5 m cuvettes together with 10 µlof0±1m riboflavin ± 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 0±1mlof1±5m (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 0±1 ml of pre-warmed 1±5m units}mg protein determined (1 unit of SOD is H#O#(for selenium-dependent activity) or 0±1mlof 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 7±4, 0±4m NAD(P)H, glutathione (reduced) for the CDNB assay, or 5 m 2±8mEDTA, 1±4mMnCl# and 0±1 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 0±1mlof10m2-mercaptoethanol and CDNB (ε ¯ 9±6m cm ) and at 360 nm for ENP −" −" the decrease in OD at 340 nm followed. One unit of (ε ¯ 0±5m 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.