INTRODUCTION
Glutathione S-transferases (GSTs) were originally described as a group of enzymes involved in the conjugation of glutathione to various metabolites in mammalian cells. They are common in the animal kingdom and are divided into different classes according to their substrate specificity (for review, see Armstrong, Reference Armstrong1991; Sheehan et al. Reference Sheehan, Meade, Foley and Dowd2001; Pearson, Reference Pearson2005). In helminths (trematodes, cestodes and nematodes) GSTs have attracted attention due to their high-level constitutive expression and antigenicity and their potential role as vaccine targets (see Mitchell, Reference Mitchell1989; Sexton et al. Reference Sexton, Milner, Panaccio, Waddington, Wijffels, Chandler, Thompson, Wilson, Spithill and Mitchell1990; Brophy and Pritchard, Reference Brophy and Pritchard1994; Barrett, Reference Barrett1995; Campbell et al. Reference Campbell, van Eldik, Liebau, Barrett, Brophy, Teesdale-Spittle and Wang2001a). Consequently, both native and recombinant enzymes have been characterized and developed for anthelmintic vaccination. GST of helminth origin are implicated in a variety of metabolic pathways by conjugation of glutathione to different agents, including detoxification of anthelmintic substances and intracellular transport (Precious and Barrett, Reference Precious and Barrett1989; Brophy and Barrett, Reference Brophy and Barrett1990a; Sharp et al. Reference Sharp, Smith, Bach, Wagland and Cobon1991; O'Leary and Tracy, Reference O'Leary and Tracy1992; Miller et al. Reference Miller, Howell and Boray1994; Kampkötter et al. Reference Kampkötter, Volkmann, de Castro, Leiers, Klotz, Johnson, Link and Henkle-Dührsen2003; Leiers et al. Reference Leiers, Kampkötter, Grevelding, Link, Johnson and Henkle-Dührsen2003). The majority of helminth GSTs characterized to date are of cytosolic origin and comprise the sigma (or alpha) class. While trematodes (first of all, Schistosoma mansoni and Schistosoma japonicum as well as the liver fluke Fasciola hepatica) were in the focus of GST-related research, nematodes have been less well characterized, with the exception of Onchocerca volvulus, a filarial pathogen of humans (for review see Gupta and Srivastava, Reference Gupta and Srivastava2006).
The advent of large-scale sequencing projects of invertebrate expressed sequence tags and whole genomes has brought about additional information regarding the GST variations of Caenorhabditis elegans, a free-living model nematode belonging to the systematic clade of the Rhabditida, and other related organisms, including members of the Strongylida (Blaxter et al. Reference Blaxter, De Ley, Garey, Liu, Scheldeman, Vierstraete, Vanfleteren, Mackey, Dorris, Frisse, Vida and Thomas1998). The Strongylida are the sister clade of the Rhabitida and include Oesophagostomum dentatum as well as other species of which native or recombinant GSTs have been characterized, such as Haemonchus contortus (Sharp et al. Reference Sharp, Smith, Bach, Wagland and Cobon1991; van Rossum et al. Reference van Rossum, Jefferies, Rijsewicjk, LaCourse, Teesdale-Spittle, Barrett, Tait and Brophy2004) and the hookworms Heligmosomoides polygyrus (syn. Nematospiroides dubius; Brophy et al. Reference Brophy, Brown and Pritchard1994a) and Ancylostoma caninum (Zhan et al. Reference Zhan, Liu, Perally, Xue, Fujiwara, Brophy, Xiao, Liu, Feng, Williamson, Wang, Bueno, Mendez, Goud, Bethony, Hawdon, Loukas, Jones and Hotez2005).
The aim of this work was to detect native GST and characterize the corresponding cDNA and genomic DNA sequences of O. dentatum and to investigate its expression during parasite development.
MATERIALS AND METHODS
Parasite material
A monospecific strain of Oesophagostomum dentatum, OD-Hann (Joachim et al. Reference Joachim, Daugschies, Christensen, Bjørn and Nansen1997), is routinely maintained by infection of parasite-free pigs kept at the animal facilities of the Institute of Parasitology and Zoology, University of Veterinary Medicine Vienna. Third-stage larvae (L3) were harvested from coprocultures and purified as described (Talvik et al. Reference Talvik, Christensen, Joachim, Bjørn, Roepstorff and Nansen1997). L3 were used as ensheathed (L3s) or exsheathed (L3x) larvae (exsheathment was performed with NaOCl as described by Joachim et al. Reference Joachim, Daugschies, Christensen, Bjørn and Nansen1997). L3x were further maintained in culture (2000 L3/ml) as described by Daugschies and Watzel (Reference Daugschies and Watzel1999) except that the standard broth was replaced by a simplified medium consisting of 90% LB-medium (20 g/l as recommended by the manufacturer (Roth, Lactan, Graz, Austria) plus 10% inactivated pig serum with antibiotics and antimycotics (i.e., 10 0000 U penicillin, 10 mg streptomycin and 2·5 μg amphotericin B per 50 ml of medium; Sigma Aldrich, Vienna, Austria). The development to fourth-stage larvae peaked after 12–14 days of cultivation when the larvae were harvested and separated by sedimentation into cultured L3 (L3c) and L4 (L4c). Post-histotropic L4 (L4p) were derived from the intestines of pigs 12 days post-infection (p.i.) and adult females (F) and males (M) were derived from the intestines of pigs during the patent stage of infection. All stages were snap-frozen in liquid nitrogen and stored at −80°C until required (for detailed methodology, see Joachim et al. Reference Joachim, Ruttkowski and Daugschies1998).
Assays of GST activity and inhibition
Homogenates of water-soluble proteins were prepared from the different stages as described by Joachim et al. (Reference Joachim, Ruttkowski and Daugschies1998) and the protein content was measured using the DC Protein Assay (Bio-Rad, Munich, Germany). These preparations were used for GST activity measurements and for GST isolation. GST activity was measured using the Glutathione S-Transferase Assay Kit (Cayman Chemical Company, Ann Arbour, USA) at 25°C for parasite homogenates from L3s, L3x, L4c, M, F, and mixed adults, and for isolated GST (supplied by the kit manufacturer). Statistical analysis was performed in Microsoft Excel (2002) using an F-test for comparison of stages.
Inhibition studies were conducted with sulfobromophthalein (SBP; dissolved in distilled water in concentrations of 0·015–1·5 mm) (SigmaAldrich, Vienna, Austria) determining the level of 50% inhibition (IC50) in L3x in relation to uninhibited controls. For bioassays (in vitro cultivation of L3x to L4c; approximately 150 L3x/well in a 24-well plate in quadruplicates) the IC50 concentrations as well as 0·1× and 10× this concentration were added to the medium during cultivation. Reversibility of the effect was evaluated in cultures which were treated for 7 days followed by a period of 7 days in SBP-free medium.
Isolation and electrophoresis of GST
GSTrap® HP columns with a volume 1 ml (Amersham Biosciences, Vienna, Austria) were loaded with whole protein preparations of L3x and eluted with reduced glutathione (Merck, Vienna, Austria) as recommended by the manufacturer. Eluates were electrophoretically separated on 12% SDS-PAGE gels and silver stained according to Blum et al. (Reference Blum, Beier and Gross1987). From the SDS-PAGE gels the separated proteins were excised and analysed by peptide digestion and mass spectrometry analysis (Max F. Perutz Laboratories, University of Vienna, Vienna, Austria).
Isolation and sequence analysis of genomic and cDNA
Genomic DNA was isolated from L3x using the PeqGold® TISSUE DNA Mini kit (PeqLab, Erlangen, Germany) as recommended by the manufacturer. Total RNA from all stages was extracted with the PeqGold® Total RNA kit (PeqLab, Erlangen, Germany) and reverse transcribed using TaqMan® Reverse Transcription Reagents (Applied Biosystems, Brunn am Gebirge, Austria) as recommended by the manufacturers.
The information obtained from peptide sequencing was compared with sequences in GenBank (using BLASTp) and partially redundant primers were designed in the conserved domains of the first and the last Exon (GST_common_FOR: 5′-CACTA(CT)AAGCT(GT)ACCTACTTC-3′ corresponding to nt 7-28 and GST_common_REV: 5′-C(CT)GG(AGT)CG(AG)GTCTC(AG)ATCCATT-3′ corresponding to nt 587-608 in Od-GST1 and nt 590-611 in Od-GST2; see below) to amplify cDNA products of 601 and 604 base pairs, respectively.
Both complementary DNA (cDNA) and genomic DNA from L3 were amplified by PCR in a volume of 50 μl using PeqGold® Taq-DNA-Polymerase (PeqLab, Erlangen, Germany) as described by the manufacturer (initially 5 min/94°C, followed by 39 cycles of 30 sec/94°C, 45 sec/56°C, 45 sec/72°C; finally 3 min/72°C). Amplicons were cloned using the TOPO® TA Cloning Kit for Sequencing (Invitrogen, Vienna, Austria) and recombinant DNA from single clones was prepared (PeqGold Plasmid MiniprepKit II; PeqLab, Erlangen, Germany) and sequenced (AGOWA, Berlin, Germany).
Based on the finding that 2 isotypes could be differentiated (see Results section), partially redundant primers were designed to amplify the 5′ ends of each isotype (FOR_GST1: 5′-ATG(GC)(CT)(AGCT)CACTA(CT)AAGCTGACC-3′; FOR_GST2: 5′-ATG(GC)(AG)(AGCT)CACTATAAGCTGACCTA-3′) in combination with a commercial anchor primer for 3′-RACE-PCR as recommended by the manufacturer (5′/3′RACE Kit, 2nd Generation, Roche Applied Science, Vienna, Austria).
Sequence analyses were either performed using the Basic Local Alignment Search Tool (BLAST; www.ncbi.nlm.nih.gov/BLAST/) or with the AlignPlus® Version of the SciEd Central Software Package for Windows (Scientific and Educational Software, Cary, NC, USA, 1999).
Expression analysis of GST isotypes
Real-time PCR of cDNA from L3s, L3x, L3c, L4c, L4p, F and M was carried out on an Mx3000p Cycler (Stratagene, La Jolla, CA, USA) using isotype-specific forward primers (5′-TAC(CT)TGGCTAGGAAATTCGG-3′ for Od-GST1 and 5′-CCTTGCGAGACAGTATGGTC-3′ for Od-GST2) and the reverse primer GST_common_REV (see above). The primers amplified a product of 399 bp in the variable region in exons 3 to 7 (see Results section) for both isotypes. As Normalizer the housekeeping gene ubiquitin-conjugating enzyme 2 (OD-ubc-2; product size: 444 bp) was amplified as described (Cottee et al. Reference Cottee, Abs El-Osta, Nisbert and Gasser2006) in 20 μl of pre-manufactured master mixes (Power SYBR® Green PCR Master Mix; Applied Biosystems, Brunamgebirge, Austria) using the same cycling conditions as above. For all stages 2 cDNA preparations were tested except L3c (1 preparation); each sample was tested in 2 independent PCR runs, each in duplicate (i.e. 6–8 replicates/stage).
RESULTS
GST activity and inhibition
The mean activity ranged from 2·27 μmol/min/mg of total protein in the L4c to 4·95 μmol/min/mg in the L3s (Table 1). The difference between these two stages was significant (P=0·0159) while the other comparisons were not (P>0·05). Concentrations of SBP >25 μm inhibited GST activity in a dose-dependent manner as shown for L3x homogenates treated with SBP in concentrations from 25 to 670 μm in 3-fold dilution steps (Fig. 1). The inhibition by 150 μm SBP was 51·8% in the isolated GST control and 62·9–68·5% in the parasite homogenates of different stages (Table 1).
Dose-dependent inhibition of GST in homogenates of L3x by SBP (n=3 replicates for each dilution). Bars indicate the minimum and maximum values, squares mark the mean values.
Activity of GST in homogenates of different stages (μmol/min/mg protein)
(Each stage was tested in 2 different batches each at least 3 times except for the separate measurements of male and female worms which were done only once and therefore not included in the statistical analysis. Inhibition was calculated in % reduction of the activity of the respective untreated control at a concentration of 150 μm SBP. Control: GST supplied by the manufacturer of the Glutathione S-Transferase Assay Kit (Cayman Chemical Company, Ann Arbour, USA).)
The development of L4 in vitro reached 18% after 14 days in the uninhibited controls. The addition of SBP in concentrations of 150 mm or 1·5 mm resulted in partial or complete inhibition of L4 development, while 15 μm SBP had no marked effect on development. When SPB was removed from the culture after 7 days, larvae resumed growth and the percentage of L4 after 14 days was comparable to the controls (Fig. 2). The larvae remained motile throughout the culture period in all groups despite growth inhibition by SBP.
Development of L4 in vitro after addition of SBP in different concentrations compared to an untreated control group (in %, average from quadruplicates). Wash: SBP was removed after 7 days by washing the larvae in SBP-free medium.
Protein analysis
SDS-PAGE, under reducing conditions, revealed 2 bands of approximately 23 and 25 kDa. Each band was excised and peptides were analysed. One peptide (KFGFAGK) was detected in both bands and analysis showed extensive homology with various other GSTs of nematode origin [mostly Chain B of C. elegans GST (GenBank Accession no. 1YQ1_B) and gst-17 and gst-37 of C. elegans (NM_064464 and NM_071131)]. Another peptide (KWIETRPASDW) was found in the 23 kDa band that had significant similarity with the C. elegans gst-38 (NM_074582).
cDNA sequence analysis
The clones derived from the amplification of the partial cDNA sequence were 73–96% similar to each other and clustered in 2 groups, Od-GST1 (601 bp) and Od-GST2 (604 bp).
Full length cDNAs were obtained using redundant isotype-specific forward primers deduced from comparisons of the obtained sequences with the 5′ ends of other nematode GSTs and 3′-RACE-PCR. The complete sequences including start and stop codon were 621 (Od-GST1) and 624 bp (Od-GST2) long with 72% overall similarityFootnote 1. The sequences also showed high similarities (73% for Od-GST1 and 70% for Od-GST2) to GST of A. caninum (GenBank Accession no. AY605283), followed by H. polygyrus GST2 (GenBank Accession no. AF128959) with 69 and 68%, and C. elegans gst-5 (GenBank Accession no. NM_063956), H. contortus GST (GenBank Accession no. AF281663), H. polygyrus GST1 (GenBank Accession no. S76129), and Ascaris suum GST (GenBank Accession no. X75502) with similarities between 57 and 63% for both isotypes (Table 2).
Pairwise comparison of nucleotide (top) and amino acid (bottom) sequence similarities (in %) between GSTs of different nematodes
(Od: Oeosophagostmum dentatum, Ac: Ancylostoma caninum, Hp: Heligmosomoides polygyrus, Hc: Haemonchus contortus, Ce: Caenorhabditis elegans, As: Ascaris suum. Data were matched over the cDNA sequences available for all GSTs. For GenBank Accession numbers see text.)
Both cDNAs consisted of 7 exons (deduced from genomic sequencing, see below) of 60–126 bp for Od-GST1 and 60–129 bp for Od-GST2, with similarities between 65 and 83% (Fig. 3).
Length and structure of the 7 exons (grey bars) and 6 introns (white bars) of Od-GST1 and Od-GST2A/B. Genomic DNA of Od-GST2 was considerably longer than that of GST2 although cDNA sequences varied only by 3 nucleotides. Within Od-GST2 two genomic structures were detected.
The proteins translated from the predicted open reading frames had 75% similarity between the two (62–95% for the different exons; Fig. 3). The similarities between Od-GST1/GST2 and other nematodes with regard to the predicted amino acids (aa) were 36–80% (Table 2; Fig. 4).
Amino acid alignments of GSTs from different nematodes deduced from the respective cDNA sequences. Od: Oeosophagostmum dentatum, Ac: Ancylostoma caninum, Hp: Heligmosomoides polygyrus, Hc: Haemonchus contortus, Ce: Caenorhabditis elegans, As: Ascaris suum. For GenBank Accession numbers of cDNA sequences see text.
The predicted sizes were around 23·57 kDa for Od-GST1 and 23·39 kDa for Od-GST-2, the calculated isoelectric points were 9·37 (Od-GST1) and 8·44 (Od-GST2). BLAST analysis (www.ncbi.nlm.nih.gov/blast/Blast.cgi) of the translated proteins revealed significant similarity to the N- and C-terminal ends of sigma-class like GSTs Conserved Domains (GenBank Accession no. cd03039 and cd03192).
Besides homologies with other nematode GSTs, protein analysis also revealed high similarity with the protein sequence of a 200 aa synthetic prostaglandin D2-synthase (GenBank Accession no. AAX37080) with 36 and 35% matching aa over the complete predicted sequences and 53% within exons 1–3 corresponding to the first 75 aa (see Fig. 3 for exon positions).
Genomic DNA sequence analysis
Primers spanning the cDNA exons 1–7 produced a product of 1296 bp for Od-GST1 and 2 sequences for Od-GST2, namely Od-GST2A with 1579 bp and Od-GST2B with 1606 bpFootnote 2. They consisted of 7 exons separated by 6 introns of varying sizes (see Fig. 3).
Expression analysis
After normalization against OD-ubc-2 (Ctubc-2 −CtGST) the ΔCt values of the different stages varied from −3·35 to 4·14 for Od-GST1 and 2·42 and 6·19 for Od-GST2 (Table 3).
Differences in threshold cycle values for quantitative RT-PCR for the two GST isoforms in different developmental stages using ubc-2 as Normalizer
(In parentheses: range of the ΔCt values between different runs. 2−ΔCt2-1: ΔCt2-1=CtOd-GST2 – CtOd-GST1.)
When L3s were used as Calibrator (as the free-living stage, in contrast to the other, parasitic, stages), the 2−ΔΔCt values indicated differential expression of the two isotypes during development; Od-GST1 was up-regulated in the parasitic stages, especially during early development of cultured L3 and L4 – corresponding to the histotropic stages – while Od-GST2 remained on lower levels with similar expression throughout development (Fig. 5).
Differences in the expression of Od-GST1 and Od-GST2 (given as 2−ΔΔCt values) in different developmental stages using ubc-2 as Normalizer and L3s as Calibrator. Values were calculated as averages from 2 independent PCR runs, each in duplicate. For all stages 2 cDNA preparations were tested except L3c (1 preparation).
In L3 the ratio Od-GST1: Od-GST2 (calculated as the 2−ΔCt2-1) was highest (1:176 for the L3s and 1:184 for the L3x), while in the advanced parasitic stages it was only 1: 2 to 1: 16 (Table 3).
DISCUSSION
Cytosolic GST activity, characterized by glutathione-binding and substrate (CDNB) metabolism, was demonstrated in the pre-parasitic and parasitic stages of O. dentatum. Based on the information from peptide sequencing of two proteins isolated by glutathione affinity chromatography and database comparison, full-length cDNA sequences for two isoforms of GST and the corresponding genomic sequences were obtained. Both consisted of 7 exons interrupted by variable introns and were expressed in increased levels in the histotropic stages of O. dentatum as determined by quantitative real-time PCR of the corresponding cDNAs. They were most similar to the GST of the canine hookworm A. caninum and belonged to the sigma-class of GSTs. They also showed significant similarity to mammalian prostaglandin D2-synthases in the conserved protein regions.
Cytosolic GSTs can be involved in a variety of cellular functions, including uptake of steroids, regulation of signal transduction pathways and anti-oxidative stress responses (as reviewed by Sheehan et al. Reference Sheehan, Meade, Foley and Dowd2001 and Mahajan and Atkins, Reference Mahajan and Atkins2005). The conjugation and subsequent inactivation of carbonyls and other metabolites of lipid peroxidation by (excretory-secretory) GSTs has been implicated as an internal and external detoxification mechanism of nematodes in the absence of other detoxification systems (Brophy and Barrett, Reference Brophy and Barrett1990b; Brophy et al. Reference Brophy, Patterson, Brown and Pritchard1995a; Pritchard, Reference Pritchard1995) primarily as a housekeeping gene function (Campbell et al. Reference Campbell, Teesdale-Spittle, Barrett, Liebau, Jefferies and Brophy2001b; van Rossum et al. Reference van Rossum, Jefferies, Young, Barrett, Tait and Brophy2001, Reference van Rossum, Jefferies, Rijsewicjk, LaCourse, Teesdale-Spittle, Barrett, Tait and Brophy2004). Activities at the host-parasite interface may also include binding of haematin and detoxification in blood-feeding H. contortus (Zhan et al. Reference Zhan, Liu, Perally, Xue, Fujiwara, Brophy, Xiao, Liu, Feng, Williamson, Wang, Bueno, Mendez, Goud, Bethony, Hawdon, Loukas, Jones and Hotez2005), the inactivation of reactive oxygen species produced by pro-inflammatory activities of the host, as suggested for the excretory-secretory GSTs of hookworms (Brophy et al. Reference Brophy, Patterson, Brown and Pritchard1995a; Pritchard, Reference Pritchard1995), or differentially expressed stress-inducible GST3 from O. volvolus (Liebau et al. Reference Liebau, Eschbach, Tawe, Fischer, Walter and Henkle-Dührsen2000; Kampkötter et al. Reference Kampkötter, Volkmann, de Castro, Leiers, Klotz, Johnson, Link and Henkle-Dührsen2003). The ability to detoxify a range of xenobiotics has been implicated in the development of anthelmintic resistance (Kawalek et al. Reference Kawalek, Rew and Heavner1984). Since GST isotypes (see below) have also been identified in the free-living C. elegans, an intrinsic function of this enzyme group is evident. However, the RNA interference experiments conducted with the C. elegans model so far have not revealed specific functions. In fact, RNAi effects could only be attributed to gst-5 which inhibits germ line apoptosis (www.wormbase.org) and, like gst-30 and gst-38, seems to be involved in oxidative stress and ageing (Ayyadevara et al. Reference Ayyadevara, Dandapat, Singh, Siegel, Shmookler, Zimniak and Zimniak2006). The finding that addition of the GST inhibitor SBP impaired the development of L4 in vitro indicates that this enzyme plays a role for nematode viability, although the underlying mechanism is yet to be elucidated.
There is growing evidence that helminth GSTs are also involved in the production of bioactive lipids (for review, see Sheehan et al. Reference Sheehan, Meade, Foley and Dowd2001). GSTs of nematode species from various clades as well as from the blood flukes S. haematobium and S. mansoni were isolated and characterized and a role in the metabolism of the anti-inflammatory prostaglandins was implicated (Liebau et al. Reference Liebau, Schönberger, Walter and Henkle-Dührsen1994; Meyer et al. Reference Meyer, Muimo, Thomas, Coates and Isaac1996; Hervé et al. Reference Hervé, Angeli, Pinzar, Wintjens, Faveeuw, Narumiya, Capron, Urade, Capron, Riveau and Trottein2003; Johnson et al. Reference Johnson, Angelucci, Bellelli, Hervé, Fontaine, Tsernoglou, Capron, Trottein and Brunori2003; Sommer et al. Reference Sommer, Rickert, Fischer, Steinhart, Walter and Liebau2003).
In O. dentatum, prostaglandins have been detected and their production could be inhibited by non-steroidal anti-inflammatory compounds (Daugschies, Reference Daugschies1995), and prostaglandins were implicated in intrinsic functions of worm biology (Daugschies and Ruttkowski, Reference Daugschies and Ruttkowski1998; Daugschies and Joachim, Reference Daugschies and Joachim2000). Although Western blots of O. dentatum homogenates showed antigenic reactivity with antibodies directed against human lipoxygenase and cyclooxygenase-2 (Joachim et al. Reference Joachim, Ruttkowski and Daugschies2001), no correlating cDNAs could be isolated (Joachim, unpublished observations). If these enzymes are absent in nematodes it is possible that, given the sequence homology between the first 75 aa of the GSTs reported here to prostaglandin D2 synthase, the GSTs may take over functions in eicosanoid metabolism. This hypothesis could explain why the GST inhibitor SBP had a negative influence on larval development of O. dentatum.
For the free-living C. elegans, 44 isoforms of GST are listed in Wormbase (www.wormbase.org). Different GST isoforms have also been characterized for A. suum (Liebau et al. Reference Liebau, Schönberger, Walter and Henkle-Dührsen1994), H. polygyrus (Brophy et al. Reference Brophy, Ben-Smith, Brown, Behnke and Pritchard1994b; Reference Brophy, Ben-Smith, Brown, Behnke and Pritchard1995b) and O. volvulus (Liebau et al. Reference Liebau, Eschbach, Tawe, Fischer, Walter and Henkle-Dührsen2000; Sommer et al. Reference Sommer, Rickert, Fischer, Steinhart, Walter and Liebau2003). Differential expression of GST isotypes has been described for H. polygyrus in relation to the immune response to the host (Brophy et al. Reference Brophy, Patterson, Brown and Pritchard1995a) and in O. volvulus in response to oxidative stress (Liebau et al. Reference Liebau, Eschbach, Tawe, Fischer, Walter and Henkle-Dührsen2000). In the present work two different isotypes of GST with considerable sequence differences could be identified, and Od-GST1 was strongly upregulated in the parasitic stages, especially in the histotropic larvae. Od-GST2, on the other hand, was downregulated in male worms. Possible explanations for the change in expression levels could be host-induced stress, changes in growth rate and metabolism or changes in the utilization of energy sources with subsequent production of endotoxic metabolites. The ratio GST2: GST1 was higher in females than in males, although not as high as in the early L3 stages. Since both males and females were derived from the same animals and treated identically, this difference may be related either to specific functions of GST in this stage or to expression of this enzyme in the maturating egg since the majority of the females was fertilized at harvesting. Changes in oxidative stress levels between the free-living and the parasitic stages can be assumed although this has not been investigated. On the other hand, the production of prostaglandins and leukotrienes in O. dentatum also occurs in a stage-specific manner (Daugschies, Reference Daugschies1995; Daugschies and Ruttkowski, Reference Daugschies and Ruttkowski1998), and a possible role of GST in this context is still to be investigated. Although a role for putative GST metabolites has been proposed in homoeostasis in lower eukaryotes (Noverr et al. Reference Noverr, Erb-Downward and Huffnagle2003), expression profiles during nematode development have not been investigated for parasitic nematodes. To date, specific functions cannot be attributed to the respective isotypes of O. dentatum GSTs; and binding kinetics to various substrates need to be investigated either for the native and the recombinant proteins, as well as expression sites and possible induction of elevated or reduced expression.
The authors thank Dr Sabine Hammer for support with the RACE PCR and R. Peschke for technical assistance with the protein work.
