Cloning and Analysis of a Surface Antigen Gene From Procyclic Trypanosoma congolense

Bayne, Rosemary Ann Lindsay (1992) Cloning and Analysis of a Surface Antigen Gene From Procyclic Trypanosoma congolense. PhD thesis, University of Glasgow.

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The presence of the variant surface glycoprotein coat, and its modulation by the process of antigenic variation have made it unlikely that a vaccine could be developed against mammalian stages of African trypanosomes. However, except for the metacyclic stage which is preadapted to life in the mammalian host, insect stages of these parasites do not possess the variant surface glycoprotein coat and display a common set of antigens on their surface dominated, in T.brucei, by a surface coat composed of the glycoprotein procyclin. If animals could be vaccinated against these common antigens then tsetse flies feeding on such vaccinated animals would ingest antibody with their bloodmeal which might inhibit development of trypanosome infections in the fly and therefore block transmission. Of a set of monoclonal antibodies which had been raised against living procyclic Trypanosoma congolense, one group appeared to be directed against molecules on the trypanosome surface and were strongly agglutinating and trypanocidal. Each monoclonal antibody in that group detected the same diffuse bands on western blots of SDS-PAGE of trypanosome lysates, indicating that they were against the same immunodominant antigen. The antigen has several unusual properties in common with procyclin and the work presented here was directed towards identifying the gene for this antigen. Heterologous probing of a genomic library with the procyclin cDNA from T.brucei indicated that T.congolense has sequences which share a degree of homology to the repetitive elements in the procyclin gene but these sequences are not expressed and there is no true homologue of this gene in T.congolense. The monoclonal antibodies did not detect the antigen gene from a cDNA expression library, probably because at least one of them appears to be directed against a carbohydrate epitope. However, differential screening of a cDNA library with first strand cDNA from procyclic and bloodstream stages detected several cDNAs, one of which contained an open reading frame with a high degree of homology to two cyanogen bromide peptide sequences derived from a Kilifi-type T.congolense surface antigen isolated by Beecroft et al (manuscript in preparation). Apart from a size difference defined by migration on SDS-PAGE, this antigen has identical properties to that detected by the set of monoclonal antibodies. The cDNA has an open reading frame coding for a protein of 256 amino acids which is rich in alanine and acidic residues. There is no N-glycosylation signal and no obvious signal peptide but the amino terminus is hydrophobic and there is a potential signal at the carboxy terminus for the addition of a glycosyl phosphatidylinositol tail which could anchor the protein in the membrane. This protein, which has been called ARP (for Alanine Rich Protein), has been expressed as a fusion protein in E.coli and used to raise antisera in rats and mice. These antisera label procyclic and epimastigote stages of T.congolense in indirect immunofluorescence and label the surface of procyclic cells in immunogold electron microscopy. In western blots of procyclic trypanosome lysates enriched for membranes they identify diffuse bands similar to those detected by the monoclonal antibodies. Several other cDNA clones were isolated in the differential screen but none of these, except perhaps cDNAPl, proved to be genuinely stage-specific. cDNAPl appears to be transcribed from telomeres but in which direction is not evident because it does not possess a poly (A) tail, although probing of northern blots suggests that some of the homologous transcripts are polyadenylated. cDNAs P7 and P8 appear to be single copy sequences in the trypanosome genome but no function can be ascribed to them as they do not contain open reading frames. cDNAPE encodes the ribosomal protein L29, is highly homologous to the the sequence of L29 from other species and may encode a cycloheximide resistant form of this protein in the stock of trypanosomes from which it was isolated. In conclusion, the apparent lack of stage-specific control at the level of transcription in trypanosomes has pushed the differential screening process to its limits and yielded sequences which are largely artifactual. (Abstract shortened by ProQuest.).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Dave Barry
Keywords: Genetics, Parasitology
Date of Award: 1992
Depositing User: Enlighten Team
Unique ID: glathesis:1992-75212
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 21:45
Last Modified: 19 Nov 2019 21:45

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