Mutations in the Aeromonas salmonicida subsp. salmonicida type III secretion system affect Atlantic salmon leucocyte activation and downstream immune responses

Deletion mutants of Aeromonas salmonicida subsp. salmonicida were used to determine the effect of the type three secretion system (TTSS) on Atlantic salmon anterior head kidney leucocytes (AHKL). One strain had a deletion in the outer membrane pore gene, ascC; and the other in three effector genes: aopO, aopH and aexT (we call this strain Δaop3). Host cell invasion success and 24h survival were depressed in ΔascC, as was 24h survival of Δaop3, when compared to the wild type strain. Challenge of AHKLs with A449 or TTSS mutants stimulated expression of the inflammatory mediators IL-8, IL-1 and TNFα at two bacterial concentrations (A600 0.1, 0.01). Expression of IL-12 was not stimulated in ΔascC challenged cells, whereas A449 and Δaop3 challenge resulted in an up-regulation of IL-12 in AHKLs, 2- and 4-fold higher than PBS, respectively. Only the wild type strain elicited a significant increase in IL-10 expression (5.5× at A600 0.1). Inducible nitric oxide synthetase (iNOS) and arginase (I+II) genes were also significantly up-regulated upon exposure to all strains. However, iNOS:arginase ratio was elevated in the effector mutant challenge. These results suggest that A. salmonicida subsp. salmonicida may enhance survival within the host cell through polarization of macrophages/leucocytes to an alternative, rather than classical, activation state. Furthermore, the short-term survival and lack of T-cell signalling cytokine stimulation in ΔascC, may help explain its inefficiency at providing protection to subsequent wild type challenge.