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Protein components of chemoreception organelles and of the cuticle of nematodes: identification and cloning of the genes, production as recombinant proteins and analysis of the immune response elicited

Objective

- To increase our knowledge and understanding, at the cellular and molecular level, of the ontogenesis, structure and physiology of chemoreception and of the cuticle of nematodes;
- To clone and study parasite molecules, components of the chemoreception system and of the cuticle, as potential target for drug and/or vaccine development;
- To study the immune response toward molecularly defined antigens of nematodes;
- To perform pilot epidemiological studies to identify, in the Northern region of Morocco, special communities or situations were nematode infections are more frequent and/or severe;
- To train scientists from European and developing countries in the field of biotechnology as applied to the control of nematode infections.

Chemoreception
- We have completely characterised a chemoreception mutant identifying a new gene dyf-1 and have shown that it affects the structure of the amphids, the main chemosensory organs of nematodes.

Avoidance mutants
- We have designed a completely new test for the chemical avoidance response. The test is relatively easy to perform, fast and unambiguous and has enabled us to screen over 150 different chemicals for the ability to trigger the avoidance reflex in C. elegans. Among the newly identified repellents are cupper ions, quinine and other antimalarial drugs. Using the assay described above, we have isolated 13 new mutants unable to avoid quinine. Some of these mutants are particularly interesting in that they apparently have normal cilia and amphidial channels. Two of these mutants do not complement and define a new gene qui-1 that we have mapped genetically on chromosome IV near the gene tra-2. The mutants fail to avoid quinine but are still able to avoid other repellents, including cupper ions and garlic extracts. This finding, together with the apparent normal architecture of the cilia, suggests that the two mutations are likely to affect the receptors directly and not the general functioning of chemosensory neurones.

Cuticle
- We have identified and cloned three homologues of cut-1 in Ascaris lumbricoides and at least two in Brugia pahangi. Southern blots with conserved regions have shown that genes homologous to cut-1 are conserved in all other nematode species tried. Together with several cut-1 like genes that have been discovered in C. elegans by the genome sequencing project they represent a new gene family with an important role in making up the protective layers of nematodes. One Ascaris gene has been studied more completely and the whole genomic and cDNA sequences determined. The homology with C. elegans cut-1 is higher than 85% as the amino acid level and like in C. elegans the protein begins with a signal peptide.
- Recombinant Ascaris CUT-1 has been obtained from E. coli expression vectors and used to raise specific antisera in rabbits. Immuno-electron-microscopy has been used to localise CUT-1 and CUT-2 epitopes in nematode cuticles. Determination of the genomic sequences and identification of cDNA clones from Brugia pahangy are in progress in Glasgow.
- Using recombinant CUT-2 produced in E. coli we have studied CUT-2 cross-linking in vitro and demonstrated the importance of hydrophobic interactions in this process, which occurs during cuticle assembly in vivo, and involves the formation of dityrosine bridges between different CUT-2 molecules.

Immune response to gp 30 and filarial antigens
- We performed a series of experiments to establish the cellular and immune response of mice to native gp30 and to a synthetic gp30 peptide that corresponds to a T cell epitope. A variety of immunisation protocols was used :
- mice immunised and boosted with L3 of Brugia pahangi;
- mice immunised sub-cutaneously with 100 micrograms of the synthetic gp30 peptide or with 100 micrograms of native Brugia antigen;
- mice infected with adult B. pahangi by transplantation into the peritoneal cavity;
- mice immunised by footpad injection with the peptide and or the adjuvant alone.

- Sera and lymphocytes from these animals were then analysed for presence of specific antibodies, for proliferation for cytokine production. Sparingly, peptide immunised animals had no detectable antibody response to Brugia antigen, while animals immunised with the native antigen or with worms did. Similarly, lymphocytes from native antigen immunised animals proliferated in response to Brugia antigen but not to peptide while cells from peptide immunised animals did not proliferate in response to native antigen but there was a modest stimulation with peptide. Finally no differences were observed in the expression of any cytokine mRNA from lymphnodes of peptide immunised animals compared to adjuvant only controls.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

INTERUNIVERSITY RESEARCH CENTRE ON DEVELOPING COUNTRIES
Address
Via Della Polveriera 37
00184 Roma
Italy

Participants (2)

INSTITUT PASTEUR DU MAROC
Morocco
Address
1,Place Charles Nicolle 1
Casablanca
NATIONAL RESEARCH COUNCIL OF ITALY
Italy
Address
Via Guglielmo Marconi 10
80125 Napoli