Analysis of virulence related determinants of Aeromonas salmonicida using transposon mutagenesis

Aquatic sciences, Animal diseases

Djebara, Mourad

University:University of Glasgow

关键词: Aquatic sciences, Animal diseases;   

Others  :  http://theses.gla.ac.uk/72862/1/10647142.pdf

来源: University of Glasgow

【 摘 要 】

Isolates of A. salmonicida were subjected to transposon mutagenesis to analyse virulence determinants produced by the organism. Initially, the work was focused on finding a suitable delivery shuttle for transposon Tn5 and preliminary results suggested that the presence of low molecular weight plasmids in A. salmonicida and/or high frequency of excision of Tn5 were the main obstacles to creating Tn5 mutants. Greater success in obtaining mutants was achieved using the suicide plasmid pRT733. Several different classes of mutants were obtained and those which failed to express the serine protease were selected for further studies. The frequency of appearance of these mutants was similar to those reported in the literature for Tn5. The main objective of this study was to create mutants lacking expression of the 70 kD serine protease, which was reported by many workers to be an important virulence factor in furunculosis in Atlantic salmon. Protease negative mutants were analysed for the expression of other potential virulence determinants such asglycerophospholipidxholesterol acyltransferase, haemolysins, the A-layer and lipopolysaccharides. Serine protease-negative mutants created by TnphoA mutagenesis exhibited low activities of the glycerophospholipid:cholesterol acyltransferase and T-lysin, factors which have been considered important in furunculosis; also, expression of the H-lysin was impared due to the lack of expression of the 70 kD protease by these mutants, thus confirming results reported by several previous workers. Analysis of caseinase-negative mutants indicated that other caseinase (s) might be expressed by the organism, thus, the role of these caseinase (s) in furunculosis should be considered. Analysis of the organisation of the A-layer in TnphoA mutants indicated that maintainance and assembly of the A-protein (the precursor of the A-layer) was closely linked to the lipopolysaccharide expressed by the organism; loss of a certain type of lipopolysaccharide, shown by loss of sensitivity to the different phages used in this study, resulted in the export of A-protein into culture fluid and disorganization of the A-layer on the surface of the organism; no loss of the A-layer was observed in these mutants. Results obtained in this study suggested that expression of the 70 kD caseinase and a certain type of lipopolysaccharide were governed by a common regulatory control sytem; also, A. salmonicida may require expression of its serine protease to attain higher levels of activities of other virulence factors which could enable the organism to invade and infect its host.