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

Virulent strains of the fish pathogen Aeromonas salmonicida, which have sur face S-layers (S+), efficiently adhere to, enter, and survive within macrop hages. 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 re constituted with exogenous S-layers, these S- mutants regained adherence. A s well, latex beads coated with purified S-layers were more adherent to fis h cell lines than uncoated beads, or beads coated with disorganized S-layer s, suggesting that purified S-layers were sufficient to mediate high levels of adherence, and that this process relied on S-layer structure. Gentamici n protection assays and electron microscopy indicated that both S+ and S- A . salmonicida invaded non-phagocytic fish cells. In addition, these fish ce lls were unable to internalize S-layer-coated beads, clearly suggesting tha t the S-layer is not an invasion factor. Lipopolysaccharide (which is parti ally exposed in S+ bacteria) appeared to mediate invasion. Surprisingly, A. salmonicida did not show net growth inside fish cells cultured in the pres ence of gentamicin, as determined by viable bacterial cell counts. On the c ontrary, bacterial viability sharply decreased after cell infection. We thu s concluded that the S-layer is an adhesin that promotes but does not media te invasion of non-phagocytic fish cell lines. These cell lines should prov e useful in studies aimed at characterizing the invasion mechanisms of A. s almonicida, but of limited value in studying the intracellular residence an d replication of this invasive bacterium in vitro.