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Site Directed Mutagenesis of the AsaP1 Toxic Petidase of Aeromonas salmonicida
J. Hentschke, Ó. H. Friðjónson, A. Ævarsson, G. Ó. Hreggviðsson and B. K. Guðmundsdóttir
Institute for Experimental Pathology, University of Iceland, Keldur v/Vesturlandsveg, 112
Reykjavík, Iceland
Aeromonas salmonicida subsp. achromogenes (Asa) infects various fish species. The
exopeptidase, AsaP1, is a major virulence factors of the bacterium. The AsaP1 encoding gene has
been cloned and expressed in E. coli and an AsaP1 negative deletion mutant of Asa has been
produced and found to have reduced virulence in salmonids and cod.
The aim of the study was to create an AsaP1 toxoid through site directed mutagenesis and test
its antigenic activity in salmon and mouse.
Amino acids, involved in substrate binding and catalytic activity of the enzyme were
identified by modeling the AsaP1 protein structure. The corresponding codons were exchanged
with site-directed mutagenesis. Glutamate in the active side of the protease was changed to
glutamine and alanine. Tyrosine in the specific binding site of the protease was changed to
phenylalanine and alanine. Casein zymograms were used to assay for enzyme activity. The
recombinant mutated proteins were injected i. p. into salmon fingerlings and pathological effects
of the proteins studied. Salmon and mice were vaccinated i.p. with the recombinant proteins and
sera were sampled eight weeks post vaccination and analysed with western blotting.
All four mutated AsaP1 proteins were found to be non-toxic to salmon. One of the mutated
enzymes was weekly proteolytic, but three showed no caseinolytic activity. The four toxoids
were found to be immunologically reactive.
SITE DIRECTED MUTAGENESIS OF THE AsaP1 EXOTOXIN OF AEROMONAS
SALMONICIDA
1
2
Institute for Experimental Pathology, University of Iceland, Keldur, Reykjavík, Iceland.
Matís ohf., Reykjavík, Iceland
Aeromonas salmonicida subsp. achromogenes (Asa) is a pathogenic bacterium, which infects
various fish species (e.g. salmonids and cod). The exoprotease, AsaP1, is one of its main
virulence factors, which induces all the pathology of atypical furunculosis when it is injected
in its pure form into salmon. The AsaP1 encoding gene has been cloned and expressed in E.
coli and an AsaP1 negative deletion mutant of Asa has been produced and found to have
reduced virulence in salmonids and cod. The aim of the study was to create an AsaP1 toxoid
through site directed mutagenesis and test its antigenic activity in salmon and mouse.
Amino acids, involved in substrate binding and catalytic activity of the enzyme were
identified by modelling the AsaP1 protein structure. The corresponding codons were
exchanged with site-directed mutagenesis. Glutamate in the active side of the protease was
changed to glutamine and alanine. Tyrosine in the specific binding site of the protease was
changed to phenylalanine and alanine. Casein zymograms were used to assay for enzyme
activity. The recombinant mutated proteins were injected i. p. into salmon fingerlings and
pathological effects of the proteins studied. Salmon and mice were vaccinated i.p. with the
recombinant proteins and sera were sampled eight weeks post vaccination and analysed with
western blotting.
All four mutated AsaP1 proteins were found to be non-toxic to salmon. One of the
mutated enzymes was weekly proteolytic, but three showed no caseinolytic activity. The four
toxoids were found to be immunologically reactive.