Anti-oxidant enzymes in Cryptosporidium parvum oocysts
13
Anti-oxidant enzymes in Cryptosporidium parvum oocysts E. E N T R A L A", C. M A S C A RO" and J. B A R R E TT#* " 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.
The role of cell-mediated immunity in host resistance to intracellular pathogens is well established (Nathan et al. 1979 ; Meshnick & Eaton, 1981 ; Murray, 1981, 1983 ; Britten & Hughes, 1986 ; Sibley, Lawson & Weidner, 1986). The antimicrobial activity of macrophages and mast cells is closely tied to oxygen radical production, triggered during phagocytosis or activation. Hydrogen peroxide, superoxide radicals and hydroxyl radicals are also generated as products of normal cellular metabolism. Cells are protected from the damaging effects of reactive oxygen intermediates by scavengers and specific enzymes such as catalase, superoxide dismutase, glutathione peroxidase and glutathione Stransferase. Little is known about the ability of parasitic protozoans to cope with reactive oxygen species generated during inflammation or when phagocytosed by macrophages. In this paper we report on the anti-oxidant enzymes in Cryptosporidium parvum. This is a protozoan which parasitizes the epithelial tissues of a wide range of vertebrates, including man, where it can cause a chronic infection in immuno-compromised individuals. Parasite material Faecal samples were collected from naturally infected newborn Holstein calves. Samples were * Corresponding author : Institute of Biological Sciences, UWA, Aberystwyth, Dyfed SY23 3DA. Tel : 01970622315. Fax : 01970-622350. E-mail : jzb!aber.ac.uk.
diluted with saline solution (0±9 % NaCl, w}v), the gross material was removed by filtration and the samples washed by centrifugation (3500 g for 15 min) and resuspended in saline solution until a low viscosity suspension was obtained. The C. parvum oocysts were purified using a caesium chloride gradient (Kilani & Sekla, 1987) and stored in saline at 4 °C.
Preparation of cell-free homogenate A number of different techniques were tried and the following gave the highest protein recovery. Purified C. parvum oocysts were excysted according to the method described by Robertson, Campbell & Smith (1993). Briefly, oocysts were suspended in Hanks balanced salt solution (HBSS) containing 1 % (w}v) trypsin and adjusted to pH 2±75 with 1 HCl. After 1 h at 37 °C the oocysts were centrifuged (2000 g for 15 min at 4 °C), resuspended in bile solution (1 % (w}v) bovine bile and 0±06 % (w}v) NaHCO in $ HBSS) and incubated at 37 °C for 30 min. Oocyst excystation and sporozoite motility was verified microscopically, before concentration by centrifugation (1500 g for 15 min at 4 °C). The pellet was 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 phosphate buffer, pH 7±2) and sonicated for 5 min with an MSE 150 Watt ultrasonic disintegrator Mk2 at 70 % power. After centrifugation at 10 000 g for 10 min at 4 °C previously washed Carborundum powder (extra fine, 300 grit, Fisons UK) was added to the precipitate and any intact oocysts were disrupted by grinding in a mortar. The viscous paste was diluted with buffer, the Carborundum powder was allowed
Parasitology (1997), 114, 13–17 Copyright # 1997 Cambridge University Press
E. Entrala, C. Mascaro and J. Barrett
to sediment and the sample was again centrifuged at 10 000 g for 10 min at 4 °C and the soluble fraction added to the previous supernatant to give a whole homogenate. The homogenate was concentrated and low molecular weight compounds (which interfered with the superoxide dismutase assay) removed by ultrafiltration in Centricon-10 microconcentrators (cut off 10 kDa, Amicon). Fractions were stored in liquid nitrogen until used. Protein was determined with Coomasie Brilliant Blue G250 (Sedmak & Grossberg, 1977) using bovine serum albumin as the standard.
Enzyme assays All assays were started by the addition of enzyme and conducted at 25 °C unless otherwise stated, concentration of reagents are final concentrations. Catalase (EC 1.11.1.6). The reaction mixture contained (in 1 ml) : 10 m H O , 50 m potassium # # phosphate buffer, pH 7±2. The decrease in OD was followed at 240 nm for 1 min. Superoxide dismutase (EC 1.15.1.1). SOD was determine by the method of Beyer & Fridovitch (1987). In this assay superoxide anions generated by the photoactivation of riboflavin reduces nitrobluetetrazolium (NBT) to an insoluble purple formazan. One ml of stock solution (containing 27 ml of 50 m potassium phosphate buffer, pH 7±8, 1±5 ml of 0±2 -methionine, 1±0 ml of 1±6 m NBT and 0±75 ml of 1 % (v}v) Triton X-100) and serial dilutions of homogenate (20–100 µl) were placed in a series of cuvettes together with 10 µl of 0±1 m riboflavin solution to initiate the reaction. After mixing, the cuvettes were illuminated for 15 min under UV light (controls without homogenate were run in parallel). The OD at 560 nm was measured before and after incubation. The rate (control divided by inhibited) was plotted against protein concentration and units}mg protein determined (1 unit of SOD is defined as the amount required to cause 50 % inhibition in the reduction of NTB). SOD activity was also determined by the method of Paoleti & Mocali (1990). This is considerably more sensitive than the NBT assay and involves the oxidation of NAD(P)H by superoxide anions generated chemically from molecular oxygen in the presence of manganous ions. The reaction mixture contained in 1 ml : 80 m triethanolaminediethanolamine buffer, pH 7±4, 0±4 m NAD(P)H, 2±8 m EDTA, 1±4 m MnCl and 0±1 ml of sample # (or buffer control). The cuvettes were mixed and the OD recorded at 340 nm for 5 min to obtain a stable baseline. The reaction was then started by the addition of 0±1 ml of 10 m 2-mercaptoethanol and the decrease in OD at 340 nm followed. One unit of
14
SOD activity is defined as the amount of enzyme required to inhibit the rate of NAD(P)H oxidation of the control by 50 %. SOD activity was also demonstrated using an on gel assay. The proteins were separated on 8–25 % native polyacrylamide gradient gels and on pH 3 isoelectric focusing gels using a Pharmacia Phastsystem. SOD activity staining was carried out with the same reagents as the NBT assay. Gels were soaked in 10 ml of the stock solution plus 0±1 ml of the 0±1 m riboflavin solution and illuminated with UV light until enzyme activity appeared as a colourless band on a blue background. To distinguish between Cu–Zn and Mn or Fe dependent SOD, activity was measured in the presence of 1 m potassium cyanide (2 m for the on gel assay) and after treatment with 5 m H O for 5 min at 37 °C # # (5 m H O , 1 m EDTA for 45 min at 37 °C for # # the on gel assay). Bovine erythrocyte superoxide dismutase together with the Mn-SOD and Fe-SOD from E. coli (Sigma) were used as positive controls. Molecular weight and pI gel markers were visualized by silver staining. Glutathione reductase (EC 1.6.4.2). The reaction mixture contained (in 1 ml) : 150 m potasium phosphate buffer, pH 7±2, 1 m EDTA, 0±14 m NADPH, 1 m glutathione (oxidized). The decrease in OD was followed at 340 nm. Glutathione peroxidase (EC 1.11.1.9). The assay was based on that described by Flohe & Gunzler (1984). The reaction mixture contained (in 1 ml) : 50 m potassium phosphate buffer, pH 7±0, 0±5 m EDTA, 0±24 units glutathione reductase (Sigma), 5 m glutathione (reduced). The mixture was allowed to equilibrate at 37 °C for 10 min, then 0±1 ml of 1±5 m NADPH in 100 m NaHCO was added and the $ peroxide-independent oxidation of NADPH followed at 340 nm for 3 min. The reaction was started by the addition of 0±1 ml of pre-warmed 1±5 m H O (for selenium-dependent activity) or 0±1 ml of # # 12 m t-butyl hydroperoxide (for non-selenium dependent activity). Glutathione S-transferase (EC 2.5.1.18). The assay was essentially as described by Habig, Pabst & Jakoby (1974) using 1-chloro-2,4-dinitrobenzene (CDNB) or 1,2-epoxy-3-(p-nitrophenoxy)propane (ENP) as substrate. The reaction mixture contained (in 1 ml) : 100 m potassium phosphate buffer, 1 m glutathione (reduced) for the CDNB assay, or 5 m for the ENP assay. The reaction was started after 2–3 min pre-incubation by the addition of 1 m CDNB or 5 m ENP. The formation of the glutathione conjugate was followed at 340 nm for CDNB (ε ¯ 9±6 m−" cm−") and at 360 nm for ENP (ε ¯ 0±5 m−" 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 , 1 m EDTA for # # 45 min at 37 °C. Lanes 1 and 2, C. parvum ; Lane 3, Cu}Zn SOD (bovine) ; Lane 4, Mn-SOD (E. coli) ; Lane 5, FeSOD (E. coli).
Trypanothione reductase. The reaction mixture contained (in 1 ml) : 100 m HEPES buffer, pH 7±2, 0±1 m EDTA, 0±5 m oxidized trypanothione, 0±25 m NAD(P)H. The decrease in OD was followed at 340 nm. Peroxidase activity (EC 1.11.1.7). This was assayed spectrophotometrically as described by Paul & Barrett (1980) using guaiacol as the substrate. The on-gel assay described by Brown, Upcroft & Upcroft (1995) was also used to assay for peroxidase using odianisidine as the substrate and for NADH and NADPH peroxidase and NADH and NADPH oxidase activities. The oocysts of C. parvum showed only low levels of SOD activity. Using the NBT assay a value of 5±7³1±4 units}mg protein (n ¯ 3) was found in dialysed samples of the soluble fraction. This activity was destroyed by heating to 100 °C for 5 min. Undialysed samples showed an activity of 22±6³2 units}mg protein but 98 % of the activity was retained after boiling for 30 min. The undialysed insoluble fraction also showed apparent SOD activity (11±4³6±4 units}mg protein), but again the activity was not destroyed by heating to 100 °C for 30 min. The apparent heat-stable SOD activity is due to the presence of free radical scavenging agents such as mannitol or alpha tocophoral. Using the NAD(P)H oxidation assay, the mean SOD activity in the dialysed soluble fraction was 53±2³4±2 units}mg protein. The higher activity with the NAD(P)H assay probably reflects the fact that this assay is less prone to interference by other cellular
components. Activity was not inhibited by 1 m KCN, but was partially inhibited by pre-incubation with 0±5 m hydrogen peroxide (23 % and 33 % after pre-incubation for 5 min and 30 min respectively) suggesting an iron-dependent SOD. From native PAGE, the molecular weight of the C. parvum SOD was estimated at 35 kDa and it appeared as a single band on isoelectric focusing gels with a pI of 4±8. The activity stain was not affected by pre-incubation of the gel with 2 m KCN, but was inhibited when the gel was pre-incubated in phosphate buffer containing 5 m H O , 0±1 m EDTA # # for 45 min at 37 °C (Fig. 1). These results are again 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 ; Becuwe et al. 1992). SOD activity in C. parvum oocysts was first described by Ogunkolade et al. (1993) as a rapidly fading band on starch gels. In the present assay, the band was stable for at least 2 months. There have been few studies on SOD from coccidia. Toxoplasma gondii has an Fe-SOD (Sibley et al. 1986), but the activity is higher than in C. parvum. In Eimeria tenella Fe-SOD only represents 30–40 % of the total SOD activity in unsporulated oocysts, copper-zinc and manganese-dependent enzymes also being present (Michalski & Prowse, 1991). In Eimeria SOD activity decreases during sporulation (from 380 to 9±8 units}mg) and only the manganese-SOD is present in fully sporulated oocysts and sporozoites. No catalase activity could be detected either spectrophotometrically or by the on-gel assay (lower limit of detection 0±5 nmoles}min}mg protein). A
E. Entrala, C. Mascaro and J. Barrett
decrease in OD at 240 nm in the presence of hydrogen peroxide caused by C. parvum extracts was found not to be destroyed by boiling and did not follow first-order kinetics. This suggests that the decrease in OD was not due to catalase, but to the direct chemical oxidation, by hydrogen peroxide, of an endogenous substrate in the parasite extract. This ‘ non-specific ’ catalase activity appeared to be associated with the insoluble fraction and it is interesting to note that Entrala et al. (1995) found that hydrogen peroxide altered the staining characteristics of C. parvum oocysts. No NADPH or NADH oxidase activity could be detected in extracts of C. parvum oocysts, nor could NADPH or NADH peroxidase activity or organic peroxidase activity be demonstrated (lower limits of detection 0±1–0±5 nmoles}min}mg protein). Glutathione transferase, glutathione reductase and glutathione peroxidase activity were not detected in C. parvum oocysts nor could trypanothione reductase activity be detected using either NADPH or NADH (lower limit of detection 0±5 nmoles}min}mg protein). However, NADPH-dependent H O consump# # tion was detected in the insoluble fraction (34±6³11±6 µmol}min}mg, n ¯ 4). Similar levels of activity have been reported in isolated late stage Plasmodium falciparum (Fairfield et al. 1988) as well as in cultured Crithidia luciliae luciliae and Crithidia luciliae thermophila (Emtage & Bremner, 1993). In these trypanosomatides NADPH-dependent H O # # scavenging systems seem to be more important than catalase, at least in response to heat stress. C. parvum lacks mitochondria. The absence of catalase and peroxidase activity and the apparent absence of glutathione and trypanothione dependent enzyme systems would suggest that there is no classical respiratory transport chain in this parasite. Oxidative protection must be being provided by alternative mechanisms such as high concentrations of free-radical scavengers such as mannitol or alternative thiols. This work was supported by a Human Capital and Mobility Grant from the EC.
, ., , ., , ., , ., , . , . (1992). Endogenous superoxide dismutase activity in two Babesia species. Parasitology 105, 177–182. , . . , . (1987). Assaying for superoxide dismutase activity : some large consequences of minor changes in conditions. Analytical Biochemistry 161, 559–566. , . , . . . (1986). American trypanosomiasis, toxoplasmosis and leishmaniasis : intracellular infections with different immunological consequences. Clinical Immunology and Allergy 6, 189–226.
16 , . ., , . . , . (1995). Free radical detoxification in Giardia duodenalis. Molecular and Biochemical Parasitology 72, 47–56. , . . , . . (1993). Thermal regulation of active oxygen-scavenging enzymes in Crithidia luciliae thermophila. Journal of Parasitology 79, 809–814, , ., -, ., , . , . (1995). Influence of hydrogen peroxide on acid-fast staining of Cryptosporidium parvum oocysts. International Journal for Parasitology 25, 1473–1477. , . ., , ., , ., , . . , . . (1988). Oxidant defense enzymes of Plasmodium falciparum. Molecular and Biochemical Parasitology 30, 77–82. , . , . . (1984). Assays of glutathione peroxidase. Methods in Enzymology 105, 114–121. , . ., , . . , . . (1974). Glutathione S-transferases. The first enzymatic step in mercapturic acid formation. Journal of Biological Chemistry 249, 7130–7139. , . . , . (1987). Purification of Cryptosporidium oocysts and sporozoites by cesium chloride and Percoll gradients. American Journal of Tropical Medicine and Hygiene 36, 505–508. , . ., , . ., , . . , . . (1984). An iron-containing superoxide dismutase in Tritrichomonas foetus. Molecular Biochemistry and Parasitology 12, 95–99. , ., , . ., , ., , . . , . (1982). Iron-containing superoxide dismutase from Crithidia fasciculata. Purification, characterisation, and similarity to leishmanial and trypanosomal enzymes. Journal of Biological Chemistry 258, 125–130. , . . , . . (1981). Leishmanial superoxide dismutase : a possible target for chemotherapy. Biochemical and Biophysical Research Communications 102, 970–976. , . . , . . (1991). Superoxide dismutase in Eimeria tenella. Molecular and Biochemical Parasitology 47, 189–196. , . . (1981). Susceptibility of Leishmania to oxygen intermediates and killing by normal macrophages. Journal of Experimental Medicine 153, 1302–1315. , . . (1983). Macrophage oxygen-dependent killing of intracellular parasites : Toxoplasma and Leishmania. Advances in Experimental Biology and Medicine 162, 127–143. , ., , ., , ., , . , . (1979). Activation of macrophages in vivo and in vitro. Correlation between hydrogen peroxide release and killing of Trypanosoma cruzi. Journal of Experimental Medicine 149, 1056–1068. , . ., , . ., , ., , . , . . (1993). Isoenzyme variation within the genus Cryptosporidium. Parasitology Research 79, 385–388. , . . , . (1980). Peroxide metabolism in the cestodes Hymenolepis diminuta and Moniezia expansa. International Journal for Parasitology 10, 121–124. , . , . (1990). Determination of
Anti-oxidant enzymes in Cryptosporidium parvum oocysts E. E N T R A L A", C. M A S C A RO" and J. B A R R E TT#* " 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.
The role of cell-mediated immunity in host resistance to intracellular pathogens is well established (Nathan et al. 1979 ; Meshnick & Eaton, 1981 ; Murray, 1981, 1983 ; Britten & Hughes, 1986 ; Sibley, Lawson & Weidner, 1986). The antimicrobial activity of macrophages and mast cells is closely tied to oxygen radical production, triggered during phagocytosis or activation. Hydrogen peroxide, superoxide radicals and hydroxyl radicals are also generated as products of normal cellular metabolism. Cells are protected from the damaging effects of reactive oxygen intermediates by scavengers and specific enzymes such as catalase, superoxide dismutase, glutathione peroxidase and glutathione Stransferase. Little is known about the ability of parasitic protozoans to cope with reactive oxygen species generated during inflammation or when phagocytosed by macrophages. In this paper we report on the anti-oxidant enzymes in Cryptosporidium parvum. This is a protozoan which parasitizes the epithelial tissues of a wide range of vertebrates, including man, where it can cause a chronic infection in immuno-compromised individuals. Parasite material Faecal samples were collected from naturally infected newborn Holstein calves. Samples were * Corresponding author : Institute of Biological Sciences, UWA, Aberystwyth, Dyfed SY23 3DA. Tel : 01970622315. Fax : 01970-622350. E-mail : jzb!aber.ac.uk.
diluted with saline solution (0±9 % NaCl, w}v), the gross material was removed by filtration and the samples washed by centrifugation (3500 g for 15 min) and resuspended in saline solution until a low viscosity suspension was obtained. The C. parvum oocysts were purified using a caesium chloride gradient (Kilani & Sekla, 1987) and stored in saline at 4 °C.
Preparation of cell-free homogenate A number of different techniques were tried and the following gave the highest protein recovery. Purified C. parvum oocysts were excysted according to the method described by Robertson, Campbell & Smith (1993). Briefly, oocysts were suspended in Hanks balanced salt solution (HBSS) containing 1 % (w}v) trypsin and adjusted to pH 2±75 with 1 HCl. After 1 h at 37 °C the oocysts were centrifuged (2000 g for 15 min at 4 °C), resuspended in bile solution (1 % (w}v) bovine bile and 0±06 % (w}v) NaHCO in $ HBSS) and incubated at 37 °C for 30 min. Oocyst excystation and sporozoite motility was verified microscopically, before concentration by centrifugation (1500 g for 15 min at 4 °C). The pellet was 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 phosphate buffer, pH 7±2) and sonicated for 5 min with an MSE 150 Watt ultrasonic disintegrator Mk2 at 70 % power. After centrifugation at 10 000 g for 10 min at 4 °C previously washed Carborundum powder (extra fine, 300 grit, Fisons UK) was added to the precipitate and any intact oocysts were disrupted by grinding in a mortar. The viscous paste was diluted with buffer, the Carborundum powder was allowed
Parasitology (1997), 114, 13–17 Copyright # 1997 Cambridge University Press
E. Entrala, C. Mascaro and J. Barrett
to sediment and the sample was again centrifuged at 10 000 g for 10 min at 4 °C and the soluble fraction added to the previous supernatant to give a whole homogenate. The homogenate was concentrated and low molecular weight compounds (which interfered with the superoxide dismutase assay) removed by ultrafiltration in Centricon-10 microconcentrators (cut off 10 kDa, Amicon). Fractions were stored in liquid nitrogen until used. Protein was determined with Coomasie Brilliant Blue G250 (Sedmak & Grossberg, 1977) using bovine serum albumin as the standard.
Enzyme assays All assays were started by the addition of enzyme and conducted at 25 °C unless otherwise stated, concentration of reagents are final concentrations. Catalase (EC 1.11.1.6). The reaction mixture contained (in 1 ml) : 10 m H O , 50 m potassium # # phosphate buffer, pH 7±2. The decrease in OD was followed at 240 nm for 1 min. Superoxide dismutase (EC 1.15.1.1). SOD was determine by the method of Beyer & Fridovitch (1987). In this assay superoxide anions generated by the photoactivation of riboflavin reduces nitrobluetetrazolium (NBT) to an insoluble purple formazan. One ml of stock solution (containing 27 ml of 50 m potassium phosphate buffer, pH 7±8, 1±5 ml of 0±2 -methionine, 1±0 ml of 1±6 m NBT and 0±75 ml of 1 % (v}v) Triton X-100) and serial dilutions of homogenate (20–100 µl) were placed in a series of cuvettes together with 10 µl of 0±1 m riboflavin solution to initiate the reaction. After mixing, the cuvettes were illuminated for 15 min under UV light (controls without homogenate were run in parallel). The OD at 560 nm was measured before and after incubation. The rate (control divided by inhibited) was plotted against protein concentration and units}mg protein determined (1 unit of SOD is defined as the amount required to cause 50 % inhibition in the reduction of NTB). SOD activity was also determined by the method of Paoleti & Mocali (1990). This is considerably more sensitive than the NBT assay and involves the oxidation of NAD(P)H by superoxide anions generated chemically from molecular oxygen in the presence of manganous ions. The reaction mixture contained in 1 ml : 80 m triethanolaminediethanolamine buffer, pH 7±4, 0±4 m NAD(P)H, 2±8 m EDTA, 1±4 m MnCl and 0±1 ml of sample # (or buffer control). The cuvettes were mixed and the OD recorded at 340 nm for 5 min to obtain a stable baseline. The reaction was then started by the addition of 0±1 ml of 10 m 2-mercaptoethanol and the decrease in OD at 340 nm followed. One unit of
14
SOD activity is defined as the amount of enzyme required to inhibit the rate of NAD(P)H oxidation of the control by 50 %. SOD activity was also demonstrated using an on gel assay. The proteins were separated on 8–25 % native polyacrylamide gradient gels and on pH 3 isoelectric focusing gels using a Pharmacia Phastsystem. SOD activity staining was carried out with the same reagents as the NBT assay. Gels were soaked in 10 ml of the stock solution plus 0±1 ml of the 0±1 m riboflavin solution and illuminated with UV light until enzyme activity appeared as a colourless band on a blue background. To distinguish between Cu–Zn and Mn or Fe dependent SOD, activity was measured in the presence of 1 m potassium cyanide (2 m for the on gel assay) and after treatment with 5 m H O for 5 min at 37 °C # # (5 m H O , 1 m EDTA for 45 min at 37 °C for # # the on gel assay). Bovine erythrocyte superoxide dismutase together with the Mn-SOD and Fe-SOD from E. coli (Sigma) were used as positive controls. Molecular weight and pI gel markers were visualized by silver staining. Glutathione reductase (EC 1.6.4.2). The reaction mixture contained (in 1 ml) : 150 m potasium phosphate buffer, pH 7±2, 1 m EDTA, 0±14 m NADPH, 1 m glutathione (oxidized). The decrease in OD was followed at 340 nm. Glutathione peroxidase (EC 1.11.1.9). The assay was based on that described by Flohe & Gunzler (1984). The reaction mixture contained (in 1 ml) : 50 m potassium phosphate buffer, pH 7±0, 0±5 m EDTA, 0±24 units glutathione reductase (Sigma), 5 m glutathione (reduced). The mixture was allowed to equilibrate at 37 °C for 10 min, then 0±1 ml of 1±5 m NADPH in 100 m NaHCO was added and the $ peroxide-independent oxidation of NADPH followed at 340 nm for 3 min. The reaction was started by the addition of 0±1 ml of pre-warmed 1±5 m H O (for selenium-dependent activity) or 0±1 ml of # # 12 m t-butyl hydroperoxide (for non-selenium dependent activity). Glutathione S-transferase (EC 2.5.1.18). The assay was essentially as described by Habig, Pabst & Jakoby (1974) using 1-chloro-2,4-dinitrobenzene (CDNB) or 1,2-epoxy-3-(p-nitrophenoxy)propane (ENP) as substrate. The reaction mixture contained (in 1 ml) : 100 m potassium phosphate buffer, 1 m glutathione (reduced) for the CDNB assay, or 5 m for the ENP assay. The reaction was started after 2–3 min pre-incubation by the addition of 1 m CDNB or 5 m ENP. The formation of the glutathione conjugate was followed at 340 nm for CDNB (ε ¯ 9±6 m−" cm−") and at 360 nm for ENP (ε ¯ 0±5 m−" 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 , 1 m EDTA for # # 45 min at 37 °C. Lanes 1 and 2, C. parvum ; Lane 3, Cu}Zn SOD (bovine) ; Lane 4, Mn-SOD (E. coli) ; Lane 5, FeSOD (E. coli).
Trypanothione reductase. The reaction mixture contained (in 1 ml) : 100 m HEPES buffer, pH 7±2, 0±1 m EDTA, 0±5 m oxidized trypanothione, 0±25 m NAD(P)H. The decrease in OD was followed at 340 nm. Peroxidase activity (EC 1.11.1.7). This was assayed spectrophotometrically as described by Paul & Barrett (1980) using guaiacol as the substrate. The on-gel assay described by Brown, Upcroft & Upcroft (1995) was also used to assay for peroxidase using odianisidine as the substrate and for NADH and NADPH peroxidase and NADH and NADPH oxidase activities. The oocysts of C. parvum showed only low levels of SOD activity. Using the NBT assay a value of 5±7³1±4 units}mg protein (n ¯ 3) was found in dialysed samples of the soluble fraction. This activity was destroyed by heating to 100 °C for 5 min. Undialysed samples showed an activity of 22±6³2 units}mg protein but 98 % of the activity was retained after boiling for 30 min. The undialysed insoluble fraction also showed apparent SOD activity (11±4³6±4 units}mg protein), but again the activity was not destroyed by heating to 100 °C for 30 min. The apparent heat-stable SOD activity is due to the presence of free radical scavenging agents such as mannitol or alpha tocophoral. Using the NAD(P)H oxidation assay, the mean SOD activity in the dialysed soluble fraction was 53±2³4±2 units}mg protein. The higher activity with the NAD(P)H assay probably reflects the fact that this assay is less prone to interference by other cellular
components. Activity was not inhibited by 1 m KCN, but was partially inhibited by pre-incubation with 0±5 m hydrogen peroxide (23 % and 33 % after pre-incubation for 5 min and 30 min respectively) suggesting an iron-dependent SOD. From native PAGE, the molecular weight of the C. parvum SOD was estimated at 35 kDa and it appeared as a single band on isoelectric focusing gels with a pI of 4±8. The activity stain was not affected by pre-incubation of the gel with 2 m KCN, but was inhibited when the gel was pre-incubated in phosphate buffer containing 5 m H O , 0±1 m EDTA # # for 45 min at 37 °C (Fig. 1). These results are again 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 ; Becuwe et al. 1992). SOD activity in C. parvum oocysts was first described by Ogunkolade et al. (1993) as a rapidly fading band on starch gels. In the present assay, the band was stable for at least 2 months. There have been few studies on SOD from coccidia. Toxoplasma gondii has an Fe-SOD (Sibley et al. 1986), but the activity is higher than in C. parvum. In Eimeria tenella Fe-SOD only represents 30–40 % of the total SOD activity in unsporulated oocysts, copper-zinc and manganese-dependent enzymes also being present (Michalski & Prowse, 1991). In Eimeria SOD activity decreases during sporulation (from 380 to 9±8 units}mg) and only the manganese-SOD is present in fully sporulated oocysts and sporozoites. No catalase activity could be detected either spectrophotometrically or by the on-gel assay (lower limit of detection 0±5 nmoles}min}mg protein). A
E. Entrala, C. Mascaro and J. Barrett
decrease in OD at 240 nm in the presence of hydrogen peroxide caused by C. parvum extracts was found not to be destroyed by boiling and did not follow first-order kinetics. This suggests that the decrease in OD was not due to catalase, but to the direct chemical oxidation, by hydrogen peroxide, of an endogenous substrate in the parasite extract. This ‘ non-specific ’ catalase activity appeared to be associated with the insoluble fraction and it is interesting to note that Entrala et al. (1995) found that hydrogen peroxide altered the staining characteristics of C. parvum oocysts. No NADPH or NADH oxidase activity could be detected in extracts of C. parvum oocysts, nor could NADPH or NADH peroxidase activity or organic peroxidase activity be demonstrated (lower limits of detection 0±1–0±5 nmoles}min}mg protein). Glutathione transferase, glutathione reductase and glutathione peroxidase activity were not detected in C. parvum oocysts nor could trypanothione reductase activity be detected using either NADPH or NADH (lower limit of detection 0±5 nmoles}min}mg protein). However, NADPH-dependent H O consump# # tion was detected in the insoluble fraction (34±6³11±6 µmol}min}mg, n ¯ 4). Similar levels of activity have been reported in isolated late stage Plasmodium falciparum (Fairfield et al. 1988) as well as in cultured Crithidia luciliae luciliae and Crithidia luciliae thermophila (Emtage & Bremner, 1993). In these trypanosomatides NADPH-dependent H O # # scavenging systems seem to be more important than catalase, at least in response to heat stress. C. parvum lacks mitochondria. The absence of catalase and peroxidase activity and the apparent absence of glutathione and trypanothione dependent enzyme systems would suggest that there is no classical respiratory transport chain in this parasite. Oxidative protection must be being provided by alternative mechanisms such as high concentrations of free-radical scavengers such as mannitol or alternative thiols. This work was supported by a Human Capital and Mobility Grant from the EC.
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