Matty Knight

Isolation and characterisation of nucleic acids from the hydatid organisms, Echinococcus spp. (Cestoda)

DNA and RNA in combination have been prepared and characterised from the hydatid disease organisms, Echinococcus granulosus and Echinococcus multilocularis. The DNA obtained is of high molecular weight, pure and can be cleaved by restriction enzymes, thereby facilitating future production of genomic DNA probes for studies of Echinococcus gene expression. Moreover, cloned DNA segments from Schistosoma mansoni hybridise strongly to Echinococcus DNA following restriction and Southern blot analysis. The extracted RNA is functional and has been translated in vitro. The major translated polypeptides and antigens have been identified, and the technique can now be used to analyse differential gene expression during development and differentiation of the hydatid organisms and to identify specific polypeptide antigens which may have potential as immunodiagnostic reagents.

Resistance to reinfection with Schistosoma haematobium in Gambian children: analysis of their immune responses

The relationship between reinfection with Schistosoma haematobium and immunological parameters was studied in a group of Gambian children aged from 8 to 13 years. Each individuals exposure to infection was assessed from observations of water contact, cercarial densities and infected snail densities at water contact sites. Eosinophil counts were made and responses to egg antigen (SEA) and adult worm antigen (WWH) measured by ELISA. Low levels of reinfection were associated with a high eosinophil count, high levels of antibodies against WWH and SEA, increased age and low exposure. In a multiple regression analysis of the association of reinfection with eosinophil count, antibody levels, exposure, age and sex, the effects of eosinophil count and exposure were still very significant after allowing for all the other variables. The effects of the antibody levels were close to significance after allowance for exposure and eosinophil count (for WWH: P = 0.09; for SEA: P = 0.07), although the evidence was less clear after additional allowance was made for age and sex. The ability of sera from the children to recognize different parasite antigens was also examined by immunoprecipitation of labelled schistosomulum surface, WWH, SEA and S. haematobium adult worm mRNA in vitro translation products. Schistosomulum surface antigens were recognized by all the sera and there was little variation in this response. There was more variation in their responses to SEA and WWH and a marked heterogeneity in the response to in vitro translation products. However, the pattern of antigen recognition appeared unrelated to susceptibility to reinfection.

Cloning of a major developmentally regulated gene expressed in mature females of Schistosoma mansoni

A cDNA library constructed from RNA isolated from adult Schistosoma mansoni has been screened by differential hybridization to identify clones corresponding to genes highly expressed by female worms. Several such cDNAs encoding the same highly abundant mRNA species were identified. Studies with one of these (pSF10) which contained a 500 base pair insert demonstrated that this gene was not expressed in immature females or eggs and encoded a polypeptide of approximately 35 000 daltons. Quantitation of the levels of RNA showed that 10% of the total RNA of female parasites was homologous to pSF10. A single gene corresponding to pSF10 was identified in Southern blotting experiments using adult worm DNA. The cloning of this gene will facilitate study of the molecular and genetic events controlling female schistosome maturation.

Repetitive DNA as a tool for the identification and comparison of nematode variants: application to Trichinella isolates.

DNA prepared from four isolates of Trichinella was compared by genomic DNA cross-hybridisation, by electrophoresis following restriction endonuclease digestion and by hybridisation studies using a cloned repetitive DNA sequence from T. spiralis. The DNA from T. spiralis, T. nelsoni and T. pseudospiralis isolates was distinct and the interrelationships of these isolates were inferred. In contrast to previous work on T. nativa and T. spiralis, our work suggests that these two isolates are very similar.

Predicted structure of a major Schistosoma mansoni eggshell protein

The complete sequence for a major Schistosome mansoni eggshell protein gene has been determined from a genomic DNA fragment. The use of an open reading frame encoding a glycine-rich polypeptide was confirmed by in vitro translation of schistosome mRNA in the presence of [3H]glycine and comparison with the amino acid composition of purified, schistosome eggshells. Apart from the extraordinary abundance of glycine and tyrosine which are evenly distributed throughout the polypeptide chain, the most striking features of the deduced amino acid sequence are the presence of five well-conserved tandem repeats of 16-18 residues in the N-terminal region and the asymmetrical distribution of charged residues. Acidic residues (Asp) are confined to the N-terminal region, while basic residues (Lys, His), with the exception of a single histidine, are found in the C-terminal region. A model structure composed of short anti-parallel beta-strands is proposed, in which glycines and residues with small side chains lie within the strands and tyrosines and cysteines are arranged at the bends, where they would be available for cross-linking. Four such strands form one of the tandem repeats which are predicted in turn to form a stack of five closely packed beta-sheets, each of three strands and linked by the more variable fourth strand. The C-terminal region may form a similar but less compact structure. The ordered structure demonstrated by birefringence studies of the schistosome eggshell [Kusel, J. (1970) Parasitology 60, 79-88] could be formed by packing of the polypeptides such that the N-terminal domain contributes counter ions or cross-links to the C-terminal domain of adjacent molecules.

A cDNA clone encoding part of the major 25000-dalton surface membrane antigen of adultSchistosoma mansoni

Immunoscreening of an adultSchistosoma mansoni cDNA expression library, using antibodies raised against purified adult worm tegumental surface membranes, identified a recombinant clone containing a 141-bp insert. Antibodies raised against the recombinant antigen bound specifically to the tegument of adult worms and immunoprecipitated the major 25000-dalton surface membrane antigen as well as a 22000-dalton nascent polypeptide generated by cell-free translation of adultS. mansoni mRNA. The mature 25000-dalton antigen was found to be precipitated by antibodies from infected mice, rats and humans.

The recognition of Schistosoma mansoni surface antigens by antibodies from patients infected with S. mansoni and S. haematobium

Polypeptide surface antigens of Schistosoma mansoni recognized by schistosomiasis patients have been identified and their strain and species specificity investigated. Antibodies from individuals infected with S. mansoni were used in immunoprecipitation assays of 125I-labelled schistosomulum surface antigens. All individuals surveyed from St. Lucia strongly precipitated antigens of approximately Mr 38,000 to 32,000 and 20,000. These antigens were shown by two-dimensional gel electrophoresis to be the same as those recognized by experimentally immunized mice. Although individuals showed a highly heterogeneous response against total polypeptide antigens synthesized in vitro by cell-free translation of adult S. mansoni mRNA, all individuals recognized the same surface antigens. Immunoprecipitation with sera from patients infected with S. mansoni in many different parts of Africa resulted in generally the same antigens being precipitated, although a very high molecular weight antigen(s), not strongly recognized by the St. Lucian sera was also precipitated by most of the African patient sera. One serum from Ghana precipitated the high molecular weight antigen but not the low molecular weight antigens, raising the possibility of the existence of S. mansoni strain(s) exhibiting some diversity in surface antigens. The surface of S. mansoni schistosomula was found to bind strongly antibodies from individuals infected with S. haematobium, demonstrating that most surface antigens are cross-reactive. Immunoprecipitation demonstrated, however, that of the polypeptide surface antigens only the very high molecular weight antigen was recognized by anti-S. haematobium antibodies and that the 38,000 to 32,000 and 20,000 Mr antigens were species-specific. Immunoprecipitation of the polypeptide antigens derived from purified adult surface membranes demonstrated recognition of the same 32,000, 25,000 and 20,000 Mr antigens recognized by chronically infected mice. Again these antigens were found to be species-specific.

Two allelic isoforms of the serotonin transporter from Schistosoma mansoni display electrogenic transport and high selectivity for serotonin.

The human blood fluke Schistosoma mansoni is the primary cause of schistosomiasis, a debilitating disease that affects 200 million individuals in over 70 countries. The biogenic amine serotonin is essential for the survival of the parasite and serotonergic proteins are potential novel drug targets for treating schistosomiasis. Here we characterize two novel serotonin transporter gene transcripts, SmSERT-A and SmSERT-B, from S.mansoni. Southern blot analysis shows that the two mRNAs are the products of different alleles of a single SmSERT gene locus. The two SmSERT forms differ in three amino acid positions near the N-terminus of the protein. Both SmSERTs are expressed in the adult form and in the sporocyst form (infected snails) of the parasite, but are absent from all other stages of the parasites complex life cycle. Heterologous expression of the two cDNAs in mammalian cells resulted in saturable, sodium-dependent serotonin transport activity with an apparent affinity for serotonin comparable to that of the human serotonin transporter. Although the two SmSERTs are pharmacologically indistinguishable from each other, efflux experiments reveal notably higher substrate selectivity for serotonin compared with their mammalian counterparts. Several well-established substrates for human SERT including (+/-)MDMA, S-(+)amphetamine, RU 24969, and m-CPP are not transported by SmSERTs, underscoring the higher selectivity of the schistosomal isoforms. Voltage-clamp recordings of SmSERT substrate-elicited currents confirm the substrate selectivity observed in efflux experiments and suggest that it may be possible to exploit the electrogenic nature of SmSERT to screen for compounds that target the parasite in vivo.

Adult schistosome cDNA libraries as a source of antigens for the study of experimental and human schistosomiasis.

Protective immunity has been demonstrated in experimental schistosomiasis and is also believed to occur in man. It can be mediated by antibodies from infected animals or animals immunized with attenuated organisms. Recombinant Escherichia coli synthesizing antigenic polypeptides from the three principal species of schistosome that infect man, Schistosoma mansoni, S. japonicum and S. haematobium, have been constructed. Libraries of adult worm cDNA were prepared from each species in the expression vector lambda gt 11 and directly screened with antibodies from animals experimentally immunized with S. mansoni and S. japonicum and from humans infected with S. haematobium. The S. mansoni clones have been analysed in greatest detail. At least four different types of clones were identified. All the detected recombinant polypeptide antigens were recognised by antibodies from chronically infected mice and most were also recognised by antibodies from mice immunized with attenuated cercariae and anti-surface membrane antibodies. Clones synthesizing species-specific antigens for both S. mansoni and S. japonicum were identified by simultaneous screening of both libraries. At least three types of S. haematobium clones were identified by screening with human infection serum, most of which were species-specific. All the antigens were in the form of fusion peptides with E. coli beta-galactosidase and their expression was induced by isopropylthiogalactopyranoside. Since known protective monoclonal antibodies recognise highly glycosylated membrane proteins which cannot be identified in the form of nascent polypeptides, the direct identification of polypeptide antigens defined by their reactivity, as reported here, is an essential step in producing reagents by recombinant DNA technology, suitable for vaccination and diagnosis.

The schistosomulum surface antigens of Schistosoma haematobium

Surface antigens of Schistosoma haematobium were identified by 125I-surface labelling of schistosomula followed by immunoprecipitation of the solubilized, labelled surfaces. The major antigens, after electrophoresis, formed a continuous smear corresponding to a molecular weight in the range 35-24 000; in addition, a 17 000 antigen was also identified. These surface antigens, in contrast to somatic antigens, were species-specific, as judged by immunoprecipitation with human anti-S. mansoni serum and serum from mice vaccinated with highly irradiated S. mansoni cercariae. S. haematobium surface antigens, however, were recognized to some extent by serum from mice chronically infected with S. mansoni. It is suggested that this cross-reactivity may reflect the heterologous immunity demonstrated experimentally between these two species, whilst the species-specificity of vaccine sera to surface antigens may mirror the highly specific immunity induced by vaccination.