Vinsamlegast notið þetta auðkenni þegar þið vitnið til verksins eða tengið í það: http://hdl.handle.net/1946/20184
Aeromonas salmonicida subsp. achromogenes is a Gram negative fish pathogen causing atypical furunculosis in Arctic charr, Salvelinus alpinus L., and several other farmed fish species. The disease can cause significant reconomic losses in the aquaculture industry. The aim of this study was to increase knowledge of the molecular virulence mechanisms of A. salmonicida subsp. achromogenes and host-pathogen interactions with focus on the toxic caseinolytic metalloendopeptidase, AsaP1, which is a major extracellular virulence factor of the bacterium and highly immunogenic. Furthermore, the aim was to develop a new vaccination strategy against atypical furunculosis based on an AsaP1-toxoid.
A. salmonicida possesses the LuxI/R type quorum sensing system, termed AsaI/R. The role of quorum sensing regulation of A. salmonicida subsp. achromogenes virulence and brown pigment production was investigated. An A. salmonicida subsp. achromogenes asaI knock-out strain was constructed by allelic insertion of a kanamycin-cassette. The mutation leads to a total knock-out of the quorum sensing system. AsaP1, cytotoxic activity, and a brown pigment secreted by the bacterium, were found to be quorum sensing regulated, but the cell associated virulence factors A-layer protein and LPS were not regulated by quorum sensing. Furthermore, the virulence of the quorum sensing knock-out strain was significantly impaired compared to the wild type (wt) strain.
A reverse transcription quantitative real-time PCR (RT-qPCR) based analysis of the innate and adaptive immune response in head-kidney, liver, and spleen of Arctic charr during a 7 d period of infection with A. salmonicida subsp. achromogenes was performed, using the wt strain and an isogenic AsaP1-deficient mutant. The results revealed an initial regulation of innate immune response parameters indicated by up-regulation of pro-inflammatory cytokine and chemokine expression. Furthermore, the immune response triggered by the two strains was significantly different, e.g., significant upregulation of a CC-chemokine was only detected in fish infected with the AsaP1-deficient mutant. This indicates the importance of AsaP1 in the development of the host defence in Arctic charr. Subsequently a Th2-cell driven adaptive immune response was detected resulting in B-cell recruitment, which was found immunohistopathologically in lymphatic white pulp surrounding splenic ellipsoid arterioles.
Four recombinant AsaP1-toxoids, AsaP1E294A, AsaP1E294Q, AsaP1Y309A, AsaP1Y309F, were constructed by single amino acid residue exchange through point mutations. The mutants AsaP1E294A, AsaP1E294Q and AsaP1Y309A were caseinolytically inactive, but enzymatic activity of AsaP1Y309F was significantly weakened compared to the wt AsaP1 enzyme. All mutants were nontoxic to fish and retained the ability to raise a specific immune response against wt AsaP1 in fish. The toxoid encoding genes, asaP1E294A and asaP1Y309F, were used for construction of two A. salmonicida subsp. achromogenes AsaP1-toxoid strains by allelic exchange. The resulting mutants secrete an AsaP1-toxoid instead of the AsaP1wt enzyme. Analysis of the recombinant AsaP1-toxoids and the A. salmonicida subsp. achromogenes AsaP1-toxoid strains revealed that AsaP1, expressed as 37 kDa pre-pro-peptide, processes autocatalytically to the mature size of 19 kDa. An experimental bacterin based on the AsaP1Y309F-toxoid strain was found to induce significant protection in Arctic charr against atypical furunculosis. The protection was comparable to protection provided by a commercial polyvalent furunculosis vaccine.
The results of this study provide new insights into the molecular regulation of AsaP1 expression, processing of the pre-pro-peptide to the mature peptidase, and the impact of AsaP1 in triggering an immune response against A. salmonicida subsp. achromogenes in Arctic charr. Furthermore,the protective capacity of an AsaP1-toxoid mutant based vaccine for fish was investigated, which is a new approach in the field of fish vaccinology.
|PhD thesis Johanna Mareile Schwenteit.pdf||3.32 MB||Opinn||Heildartexti||Skoða/Opna|