DEFINED DELETION MUTANTS DEMONSTRATE THAT THE MAJOR SECRETED TOXINS ARE NOT ESSENTIAL FOR THE VIRULENCE OF AEROMONAS-SALMONICIDA

The importance of the two major extracellular enzymes of Aeromonas sal monicida, glycerophospholipid: cholesterol acyltransferase (GCAT) and a serine protease (AspA), to the pathology and mortality of salmonid f ish with furunculosis hail been indicated in toxicity studies. In this study, the genes encoding GCAT (satA) and AspA (aspA) have been clone d and mutagenized by marker replacement of internal deletions, and the constructs have been used for the creation of isogenic satA and aspA mutants of A. salmonicida. A pSUP202 derivative (pSUP202sac) carrying the sacks genes was constructed to facilitate the selection of mutants . The requirement of serine protease fear processing of pro-GCAT was d emonstrated. Processing involved the removal of a short internal fragm ent, Surprisingly, pathogenicity trials revealed no major decrease in virulence of the A. salmonicida Delta satA::kan or A. salmonicida Delt a aspA::kan mutants compared to the wild-type parent strains when Atla ntic salmon (Salmo salar E.) were challenged by intraperitoneal inject ion. Moreover, using a cohabitation model, which more closely mimics t he natural disease, there was also no significant decrease in the rela tive cumulative mortality following infection with either of the delet ion mutants compared to the parent strain. Thus, although these two to xins may confer some competitive advantage to A. salmonicida, neither toxin is essential far the very high virulence of A. salmonicida in At lantic salmon. This first report of defined deletion mutations within any proposed extracellular virulence factor of A. salmonicida raises c rucial questions about the pathogenesis of this important fish pathoge n.