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

Abstract

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.

Phenotype of Aeromonas salmonicida sp. salmonicida cyclic adenosine 3′,5′‐monophosphate receptor protein (Crp) mutants and its virulence in rainbow trout (Oncorhynchus mykiss)

“…The adhesion data showed that A. salmonicida ∆ crp (J250) attached to CHSE‐214 cells (non‐phagocytic cells) in a similar fashion as the wild‐type strain J223 (Figure a). On the other hand, the invasion experiment, where gentamicin was used to differentially kill extracellular bacteria (Garduño et al., ), indicated that A. salmonicida ∆ crp has an increased intracellular invasion rate on CHSE‐214 cells ( p < .0012) versus the wild‐type strain J223 (Figure b). The viability of the CHSE‐214 cells has no significant changes during the assay (Figure c).…”

“…Common bluegill ( Lepomis macrochirus ) embryo cells (CHSE‐214) were grown at 15°C in Eagle's minimum essential medium (Corning Incorporated, NY, USA) supplemented with 10% of heat‐inactivated FBS (Corning Incorporated) and 1% MEM non‐essential amino acids (Corning Incorporated) without antibiotics (Garduño et al., ).…”

The involvement of Aeromonas salmonicida virulence factors in bacterial translocation across the rainbow trout, Oncorhynchus mykiss (Walbaum), intestine

“…Exposure of epithelia to this endotoxin may cause over‐activation of the immune system resulting in inflammation that may be harmful and even fatal to the host (Munford 2005). Adhesion proteins are found in the S‐layer covering the outer surface of the bacteria as well as in the form of protruding type IV pili (Garduno, Moore, Olivier, Lizama, Garduno & Kay 2000; Masada, LaPatra, Morton & Strom 2002). However, although some of the effects of the virulence factors from A. salmonicida have been determined at the whole animal level, focus has not been directed towards determining their possible role during the initial stage of infection, i .…”

Polyphasic characterization of Aeromonas salmonicida isolates recovered from salmonid and non‐salmonid fish

“…Such genetic and phenotypic differences among our isolates may be associated with altered pathogenicity, as has been observed for A‐layer protein array of A. salmonicida in other studies (Kay & Trust ; Garduno et al . ).…”