Peter M. Brophy

Immunity to helminths: Ready to tip the biochemical balance?

Using data from recent laboratory and field studies, Peter Brophy and David Pritchard here summarize our current understanding of the approximate sequence of events leading to protective immune responses against some chronic parasitic helminth infections.

BIOCHEMICAL ANALYSIS, GENE STRUCTURE AND LOCALIZATION OF THE 24 KDA GLUTATHIONE S-TRANSFERASE FROM ONCHOCERCA VOLVULUS

Survival of Onchocerca volvulus, a pathogenic human filarial parasite, is likely to depend upon the detoxification activities of the glutathione S-transferases (GSTs). The 24 kDa O. volvulus GST, OvGST2, was expressed in a bacterial system and the recombinant protein was purified to homogeneity by affinity chromatography. Specific activities of the recombinant OvGST2 (rOvGST2) with a variety of substrates, and in the presence of inhibitors, were determined. With the universal substrate 1-chloro-2,4-dinitrobenzene, the specific activity of rOvGST2 was 2130 nmol min-1 mg-1. The rOvGST2 showed relatively limited selenium-independent glutathione peroxidase activity, but secondary products of lipid peroxidation, namely members of the trans,trans-alka-2,4-dienal,trans-alk-2-enal and 4-hydroxyalk-2-enal series, were conjugated to glutathione via OvGST2 dependent activity. The gene encoding the OvGST2 was isolated and the nucleotide sequence determined. The ovgst2 gene was found to possess seven exons with six intervening sequences, with all except one having consensus splice-site junctions. This intron/exon organisation of the ovgst2 gene is almost identical with those described for the mammalian Pi class GST genes, consistent with the protein structural evidence that the OvGST2 is related to the Pi class GSTs. Southern blot analysis with total parasite genomic DNA indicated a single copy gene, with a restriction pattern consistent with that of the isolated gene. The tissue distribution of the OvGST2 was examined in O. volvulus by immunohistochemistry and was shown to be distinct from that of the OvGST1. The OvGST2 was located throughout the syncytial hypodermis of male and female adult worms, as well as in the uterine epithelium. Microfilariae, and infective third stage larvae of O. volvulus, isolated from Simulium neavei, were immunopositive for OvGST2.

Glutathione S-transferase (GST) expression in the human hookworm Necator americanus: potential roles for excretory-secretory forms of GST

The difficulty in demonstrating protective immunity to human gastro-intestinal nematodes is thought to be a consequence of the expression of defences by the parasites directed against the toxic metabolites of leukocytes produced during inflammation (Brophy and Pritchard, 1992a). Parasite glutathione S-transferases (GSTs) may provide part of this defence by detoxifying the secondary products of lipid peroxidation produced via immune initiated free-radical attack on host or parasite membranes (Brophy and Pritchard, 1994; Taylor et al., 1988). Neutralisation of parasite immune defence components could tip the molecular balance in favour of the immune response during chronic infections. For example, GSTs have been extensively investigated from the digenean parasites Schistosoma and Fasciola hepatica and provide protection in animal-model systems (Mitchell, 1988; Wijffels et al., 1991). In contrast, although GSTs have been initially characterised in filarial nematodes (Salinas et al., 1994; Leibau et al., 1994; Jaffe and Lambert, 1986), there is limited information on GSTs from human gastro-intestinal nematode parasites. We were particularly interested in analysing the products of hookworms for evidence of the presence of excretory-secretory forms of this putative immune defence protein.

Metabolism of lipid peroxidation products by the gastro-intestinal nematodes Necator americanus, Ancylostoma ceylanicum and Heligmosomoides polygyrus

Somatic extracts of the three parasitic nematodes Necator americanus, Ancylostoma ceylanicum and Heligmosomoides polygyrus were able to detoxify a model hydroperoxide and a putative natural peroxide by glutathione-dependent peroxidase activity while cytotoxic carbonyls could be metabolized by NADPH-linked reduction activities. Unlike cestodes and digeneans, the nematodes in this study could not enzymatically conjugate carbonyls with glutathione. The results indicate that the three nematodes can protect themselves against possible host-immune initiated lipid peroxidation of their membranes at the level of the hydroperoxide and at the level of cytotoxic carbonyl, although other protective enzymatic mechanisms are also likely to exist (superoxide dismutase and catalase).

Glutathione S-transferases from the gastrointestinal nematode Heligmosomoides polygyrus and mammalian liver compared

Glutathione S-transferases have been partially characterised from the gastrointestinal nematode Heligmosomoides polygyrus. Two major subunit families were purified (24 and 23 kDa) with N-terminal homology to the mammalian Alpha family. Four dimeric forms of GST were purified from the nematode by glutathione-affinity chromatography, two major enzymes (pI 8.1, 5.0) and two minor forms (pI 5.8, 5.3). The purified GST pool could neutralize model and lipid peroxides via peroxidase activity but not peroxidation derived reactive carbonyls via glutathione transferase activity. Antisera raised to the pooled nematode GSTs appeared to recognize other Strongylida GSTs more strongly on Western blotting compared to mammalian GSTs.

Differential expression of glutathione S-transferase (GST) by adult Heligmosomoides polygyrus during primary infection in fast and slow responding hosts

Glutathione S-transferase (GST) specific enzymatic activity, assayed with the model substrate 1-chloro-2,4-dinitrobenzene, was 45% higher in adult Heligmosomoides polygyrus passaged through a slow responder mouse strain, C57/BL10 compared to worms passaged through a fast-responder strain (SWR x SJL) F1. Western analysis using polyclonal antisera raised to purified H. polygyrus GSTs did not appear to positively correlate the expression of GST protein with functional enzymatic activity. However, western blotting did indicate a sex-linked expression pattern of GST protein, with male worms expressing a higher ratio of the 24 kDa to the 23 kDa GST family than female worms.

A PCR strategy for the isolation of glutathione S-transferases (GSTs) from nematods

Anchor based PCR technology has been used to isolate a GST sequence from the gastro-intestinal nematode Heligmosomoides polygyrus. A 800 base pair product was amplified from first-strand cDNA using primers based on the N-terminal sequence of purified H. polygyrus GST (upstream primer) and a non-specific polyadenylate tail with an anchor sequence (downstream primer). The product was cloned into pUC18 and sequenced. A reading frame of 648 bases in the sequence encoded a protein which has 30% homology with the alpha family of mammalian glutathione S-transferases.

Comparative metal content profiling of parasitic helminths by electron paramagnetic resonance spectrometry: significance for metalloprotein content.

Variation in co-ordination geometries of metal ions bound to proteins imposes electronic states different from free (hydrated) ions in solution. Electron paramagnetic resonance spectroscopy has been used to analyse a selection of parasitic helminths for metal content as an initial step to determination of metallo-enzymes in their ES products under immune stress conditions. Characteristic paramagnetic resonance spectroscopy spectra show clear evidence for the presence of iron, copper, and manganese centres and in the selected parasites. The metals ions are identified as protein-bound as distinct from free metal ions present in aqueous solution, and distinguishable from parasite dietary components derived from host sources. Indication is given that superoxide dismutases may, in part, account for the metal ions observed. The use of electron paramagnetic resonance spectroscopy to identify specific protein-bound metals without prior isolation of the suspected protein is here applied.

Glutathione binding proteins in the gastrointestinal nematode Heligmosomoides polygyrus.

A glutathione-affinity matrix was used to identify glutathione-dependent protein(s) in somatic extracts of the nematode Heligmosomoides polygyrus. Polypeptides of 70-80 kDa were retained by the affinity matrix following the elution of H. polygyrus glutathione S-transferases by 5 mM glutathione. The 70-80-kDa polypeptides were subsequently eluted from the matrix by the addition of 20 mM glutathione and these polypeptides did not show glutathione S-transferase activity. The high-affinity H. polygyrus glutathione-binding proteins may be related to the uncharacterized purification hindering factors previously demonstrated during the isolation of glutathione S-transferases in several other helminths.

The Production and Analysis of Helminth Excretory-Secretory (ES) Products

Parasitic helminths secrete and excrete a wide range of molecules during culture in vitro. Given the limitation of these culture conditions, one must presume that the molecules secreted have a biological function, and a role to play in maintaining the homeostasis of the host/parasite relationship. In the present chapter we have attempted to review the conditions already used to maintain a range of parasites considered to be of medical, economic and biological importance. In doing so, we have also attempted to draw the attention of the reader to some of the more chemically defined molecules found in culture supernates or excretory-secretory (ES) products. Particular importance has been placed on molecules considered to be essential to the maintenance of the life cycle of these parasites, as these molecules probably represent an “Achilles heel” of many of the parasite species described.