Host cell invasion and intracellular residence by Aeromonas Salmonicida: Role of the S-layer

Virulent strains of the fish pathogen Aeromonas salmonicida, which have surface S-layers (S⁺), efficiently adhere to, enter, and survive within macrophages. Here we report that S⁺ bacteria were 10- to 20-fold more adherent to non-phagocytic fish cell lines than S-layer-negative (S^-) mutants. When reconstituted with exogenous S-layers, these S-mutants regained adherence. As well, latex beads coated with purified S-layers were more adherent to fish cell lines than uncoated beads, or beads coated with disorganized S-layers, suggesting that purified S-layers were sufficient to mediate high levels of adherence, and that this process relied on S-layer structure. Gentamicin protection assays and electron microscopy indicated that both S⁺ and S^- A. salmonicida invaded non-phagocytic fish cells. In addition, these fish cells were unable to internalize S-layer-coated beads, clearly suggesting that the S-layer is not an invasion factor. Lipopolysaccharide (which is partially exposed in S⁺ bacteria) appeared to mediate invasion. Surprisingly, A. salmonicida did not show net growth inside fish cells cultured in the presence of gentamicin, as determined by viable bacterial cell counts. On the contrary, bacterial viability sharply decreased after cell infection. We thus concluded that the S-layer is an adhesin that promotes but does not mediate invasion of non-phagocytic fish cell lines. These cell lines should prove useful in studies aimed at characterizing the invasion mechanisms of A. salmonicida, but of limited value in studying the intracellular residence and replication of this invasive bacterium in vitro.