A number oi R.salmoninarum gene libraries were constructed in E.coli host-vector systems and screened for the production of molecules which may provide material for the immunisation of salmonid fish. One immunopositiye clone was isolated from a £coRI gene bank constructed in the plasmid vector pUC18 and another clone, which possessed membrane-active properties, was isolated from a M«dIII gene bank constructed in the plasmid vector pBR328. The immunopositive clone was found to contain the major portion of gene msa encoding the major secretory antigen of R. salmoninarum, known as P57. The membrane-active clone possessed a broad activity against erythrocytes from a number of mammalian species and rainbow trout, but not rabbits. No lecithinase, caseinase or gelatin degrading activities were detected in active clones. The haemolytic product was not identifiable on either SDS-PAGE gels or Western blots of cells taken from the active clone. However, minicell analysis revealed that the membrane-active protein was about 65K, and the promoter region of the gene encoding this protein was identified. Southern blotting showed that the gene was present in seven strains of R.salmoninarum of differing virulence. The gene was sequenced and the sequence analysed by computer. After comparison with the contents of the Protein Identification Resource database, the gene was found to encode a protein which bears strong similarities with a family of secreted zinc-dependent metalloendopeptidases and on this basis the gene was tentatively named mpr and the product, MPR. Aspects of the predicted structure and possible function of MPR are discussed. In order to simplify the purification of material for further studies, gene fusions were constructed, from msa, mpr and hly, a gene encoding another membrane-active product from R.salmoninarum, in either pMAL or pAX5 vectors. The resultant fusion proteins were produced in a mainly soluble form and purified by affinity chromatography. The fusion proteins were found to be immunogenic in rats but only poorly inuuunogenic in rainbow trout. Epitopes of each of the fusion proteins were identified on Western blots by serum derived from rainbow trout undergoing a clinical outbreak of BKD and it is proposed that this provides circumstantial evidence that the native molecules are of immunological importance to BKD and may be usefiil candidates for future immunisation studies. Seven strains of R.salmoninarum were cultured in vitro under conditions of either ironrestriction or iron-sufficiency. Epitopes of each of the fusion proteins were identified in R.salmoninarum cell extracts and, in the case of msa, in the ECP. There is preliminary evidence that the production of MPR and the processing of P57 may be regulated by the availability of iron or other metals. No evidence for the production of siderophores by R.salmoninarum was found, although culture supernatants did possess some ability to inhibit the binding of iron by transferrin. A strong iron reducing activity was found to be associated with R.salmoninarum cells and the production of reducing sugars accompanied iron-restricted culture conditions. Comparative discussion of the pathogenicity of R.salmoninarum and of other Gram positive intracellular pathogens is provided.

Document Type


Publication Date