Echinococcus granulosus: Heterogeneity and Differential Expression of Superoxide Dismutases

Experimental Parasitology 94, 56–59 (2000) doi:10.1006/expr.1999.4464, available online at http://www.idealibrary.com on

RESEARCH BRIEF Echinococcus granulosus: Heterogeneity and Differential Expression of Superoxide Dismutases

G. Salinas and S. Cardozo Ca´tedra de Inmunologı´a, Instituto de Higiene, Avda. A. Navarro 3051, Montevideo, Uruguay

as the hydroxyl (?OH), hydrodioxyl (HO2?), and peroxynitrite (ONOO?) radicals. Eukaryotes possess two types of SOD: MnSOD, which is found in the mitochondria, and CuZnSOD, localized in the cytosol, peroxisome, or extracellular fluids (Fridovich 1995). The importance of these enzymes in parasitic organisms has been highlighted by several reports in which resistance to oxidative damage has been correlated with the level of antioxidant enzymes (James 1994). Although nematode and trematode SODs have been extensively characterized at the biochemical and molecular levels (LoVerde 1998; Selkirk et al. 1998), very few studies have been reported on cestode SOD. A CuZnSOD has been partially characterized from Taenia taeniaeformis (Leid and Suquet 1986), and SOD activity has been reported in Echinococcus granulosus in a study of genetic variation of parasite isolates from different hosts and geographical regions (Lymbery and Thompson 1988). We have started to characterize the antioxidant enzymes of E. granulosus, the causative agent of hydatid disease. While thioredoxin peroxidase may be the main hydrogen peroxide-metabolizing enzyme of this organism (Salinas et al. 1998), the results presented in this article suggest that CuZnSOD activity is developmentally regulated in E. granulosus and actively secreted by protoscoleces. All E. granulosus extracts assayed possessed CuZnSOD activity (Table I), which was highest in protoscoleces (PSC). MnSOD activity was detected only in PSC and prepatent adult worm extracts, constituting less than 15% of the total SOD activity in either case. The specific activity in PSC E/S products was about five times greater than that of the extract, suggesting that CuZnSOD is actively secreted in vitro by PSC. Low levels of CuZnSOD activity were detected in hydatid fluid (HF), indicative of in vivo secretion of the enzyme, and in hydatid cyst wall (HCW). An interesting observation was that extracts of HCW and HF from nonfertile cysts did not possess detectable levels of enzyme activity. To further characterize the activity, different parasite extracts were resolved by native PAGE and stained for SOD activity. Five clear bands of activity were present in PSC extracts (Fig. 1A). Inhibition

Salinas, G. and Cardozo, S. 2000. Echinococcus granulosus: Heterogeneity and differential expression of superoxide dismutases. Experimental Parasotology 94, 56–59. q 2000 Academic Press Index Descriptors and Abbreviations: Echinococcus; cestode, hydatid disease; ROS, reactive oxygen species; SOD, superoxide dismutase; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; NBT, nitroblue tetrazolium; PBS, phosphate-buffered saline; HF, hydatid fluid; HCW, hydatid cyst wall; PSC, protoscolex.

Reactive oxygen species (ROS) are an inescapable consequence of aerobic metabolism: the sequential reduction of oxygen to water produces superoxide radical and hydrogen peroxide. These species may have potentially deleterious effects, since they can interact with each other and with cellular components, leading to radical chain reactions and ultimately to cell death. Antioxidant enzymes constitute a natural regulatory system which has evolved to enable aerobic organisms to employ oxidative metabolism while being protected from the damaging effects of its by-products (Halliwell and Gutteridge 1999). Parasitic organisms have an additional requirement for these enzymes, since they may also be exposed to oxidizing agents derived from host effector cells (Callahan et al. 1988; LoVerde 1998; Selkirk et al. 1998). It is thought that they have adapted to this stress by synthesizing high levels of antioxidant enzymes and expressing them at the host–parasite interface; for instance, several variants of helminth antioxidant enzymes are localized to the tegument or the cuticle and are released into their immediate environment (Cookson et al. 1992; Tang et al. 1995; Mei and LoVerde 1997; Liddell and Knox 1998). Superoxide dismutases play a key role as antioxidant enzyme by catalyzing the dismutation of the superoxide radical to hydrogen peroxide and molecular oxygen. Although superoxide is not extremely harmful per se, it can lead to the formation of highly toxic species, such

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E. granulosus SUPEROXIDE DISMUTASE

TABLE I SOD Activity in Different E. granulosus Extracts Extract Aqueous soluble PSC extract E/S PSC products Hydatid fluida Hydatid cyst wall extracta Prepatent adult worm extract

Total SOD activity MnSOD activity (U/mg) (U/mg) 35 148 3 5 10

5 ND ND ND 1

Note. Activity was determined according to McCord and Fridovich (1969), in the absence or presence of 1 mM KCN (total and MnSOD activity, respectively). Values represent mean of triplicate assays. Protoscoleces (PSC) and hydatid fluid (HF) were obtained by aseptic puncture of hydatid cysts. Extracts were prepared by homogenization on liquid nitrogen in PBS, pH 7.6, containing protease inhibitors, followed by centrifugation at 15,000g for 30 min. HF was supplemented with EDTA to a final concentration of 5 mM and concentrated 10 times by ultrafiltration. PSC E/S products were obtained by incubating 1 ml packed vol PSC in 5 ml of RPMI 1640, pH 7.5, containing 1% glucose and 2 mM glutamine at 378C for 2 h. Protein concentration was determined using the bicinchoninic acid protein assay kit (Pierce, U.S.A.). Interference with glucose in this assay was eliminated by ultrafiltration of E/S products with centricon YM3. ND, nondetectable. a HF and HCW extract values from fertile hydatid cysts. Extracts from nonfertile cyst did not possess detectable levels of SOD activity.

with KCN identified the form with slowest electrophoretic mobility as MnSOD and the four faster migrating bands as CuZnSOD (Fig. 1B). These four CuZnSOD activity bands were also present in PSC E/S products and HF, indicating that they are secreted in vivo. In accordance with our results, T. taeniaeformis CuZnSOD, the only other SOD characterized from a cestode parasite, has been reported to be present in the cyst fluid (Leid and Suquet 1986). Prepatent adult worm

extracts showed two bands of CuZnSOD activity with fast electrophoretic mobility, while the HCW extract showed one band of CuZnSOD activity, with slower electrophoretic mobility than those of PSC. The electrophoretic profile of SOD activity in different stages and preparations suggest that E. granulosus contains several isoforms, whose expression is developmentally regulated. This phenomenon has also been documented in nematodes (Ou et al. 1995; Wildenburg et al. 1998) and trematodes (Hong et al. 1992; Mei and LoVerde 1997; Piacenza et al. 1998). Although the hydatid cyst provides physical protection to the parasite, this can be damaged by the immune system, especially before formation of the collagenous capsule. Thus, inactivation of effector mechanisms is probably necessary for parasite survival. Protection against superoxide release by host cells at the metacestode interface could be achieved by SOD present in the cyst wall and hydatid fluid. The fluid bathes the inner germinal layer of the cyst, containing the live parasite, which gives rise to the PSC toward the inner face. In this context it is interesting to note that nonfertile cysts, usually associated with more pronounced chronic inflammatory reactions and degeneration of the parasite (Rao and Mohiyuddin 1974; Thomas and Kothare 1975; reviewed in Thompson 1995), did not contain detectable levels of SOD activity, either in their HCW extracts or HF. The high level of SOD expression by the PSC and secretion into the immediate environment would not only be beneficial for protection of the cyst germinal layer but also might be important for infectivity of PSC in the definitive host. The larval worm is, once attached to the dog gut, in close contact with the submucosa, and the scolex of the developing worm is in intimate contact with the systemic circulation (reviewed in Thompson 1995). Adult worm extracts also showed moderate levels of SOD activity, with a different electrophoretic profile than that of PSC. It would be interesting to follow the pattern of expression during differentiation toward the adult worm; the lack of a model for E. granulosus dog infection precluded this study. The CuZnSOD activity from E. granulosus PSC was purified from the total aqueous extract by ammonium sulfate precipitation, anion exchange, and chelating chromatography. SOD isoforms eluted sequentially from the anion exchange column at 80–120 mM NaCl (Fig. 2A).

FIG. 1. NBT–PAGE from E. granulosus extracts. Different extracts of E. granulosus were fractionated under native conditions on 10% PAGE according to Laemmli (1975) and stained for SOD activity according to Beauchamp and Fridovich (1971) in the absence or presence of 1 mM KCN (A and B, respectively). Lanes 1 and 2, hydatid cyst wall extracts from fertile and nonfertile cysts, respectively; 3, hydatid fluid from fertile cysts; 4, protoscolex E/S products; 5, protoscolex extract; 6, prepatent adult worm extract.

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FIG. 2. Purification of CuZnSOD from E. granulosus protoscoleces (PSC). The aqueous soluble fraction of homogenized PSC was fractionated by precipitation between 55 and 90% saturated ammonium sulfate. This fraction was dialyzed against 40 mM Tris–HCl, 10 mM NaCl, pH 8.5, and chromatographed on a Mono Q HR 10/10 column using a continuous 10–500 mM NaCl gradient. Fractions were analyzed by native NBT–PAGE (A: lane 1, total protoscolex extract; lanes 2–7, sequential eluates from 80 to 120 mM NaCl). Fractions containing CuZnSOD activity were pooled and applied to a Chelating Superose HR 10/2 column, previously saturated with CuSO4. Elution was performed with a continuous 0–50 mM imidazol gradient. All activity bands eluted in one fraction at 17 mM imidazole (B). This fraction was analyzed by SDS–PAGE under reducing conditions (C). The position of molecular weight markers is shown in kDa.

Active fractions were pooled and fractionated by chelating Sepharose chromatography, all the isoforms eluting with 17 mM imidazole (Fig. 2B). The specific activity of this fraction was 910 U/mg; thus, the purification scheme resulted in a 30-fold SOD enrichment (see Table I). Silver staining after SDS–PAGE of this fraction showed the presence of proteins at 18 and 37 kDa (Fig. 2C). T. taeniaeformis SOD has been shown to have an apparent Mr of 16.6 kDa on SDS–PAGE under reducing conditions (Leid and Suquet 1986). The 37-kDa protein band observed in the purified fraction from PSC extract may thus represent a nondissociable dimer or a monomer corresponding to a different isoform. The possibility of this being a contaminant protein cannot be completely ruled out, but it seems unlikely since silver and SOD staining of native gels revealed protein and enzyme activity bands at the same electrophoretic mobility (data not shown). We are currently pursuing N-terminal sequence analysis of both protein bands in order to isolate cDNA clones and further characterize E. granulosus CuZnSOD.

the International Centre for Genetic Engineering and Biotechnology (Italy). We are grateful to Dr. M. Selkirk and Dr. C. Ferna´ndez for helpful discussions of the manuscript.

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This work was supported by research grants from the Wellcome Trust (UK), the International Foundation for Science (Sweden), and

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