Free-living amoebae are gaining increasing attention as ubiquitous eukaryotes influencing our perception of human bacterial pathogens in the environment. For example, the facultatively intracellular mammalian pathogens Legionella pneumophila
and mycobacteria are also known to prosper within free-living amoebae, implying that such bacteria are adapted to an intracellular environment in a more general sense (32
). Furthermore, L. pneumophila
is known to use the same sets of genes for multiplying in human macrophages and A. castellanii
Salmonellae are rather promiscuous in their ability to invade and replicate in mammalian host cells. In contrast, the interaction between salmonellae and Acanthamoeba
species has not been described in any detail. In the present study we observed a preferential uptake of Salmonella
serovar Dublin over that of serovar Enteritidis or serovar Typhimurium in all isolates of Acanthamoeba
tested (Fig. ). Since by definition the bacterial serovars express different types of sugar entities in the surface O-antigen polysaccharide, one may suggest that this results in differential recognition by the amoebae and hence in different efficiencies of uptake mediated by the lectin-like receptors on the surfaces of the amoebae. We also found that bacterial growth conditions had significant effects on the efficiency of entry into amoebae. Broth (LB) cultures were more efficiently taken up by A. rhysodes
than were bacteria propagated on solid (LA) medium. It is possible that this significant difference in uptake can be related to the different entry mechanisms used. Entry of salmonellae into mammalian host cells, especially epithelial cells of the gut, depends on virulence factors that are encoded by SPI1 and are induced under LB growth conditions (12
). However, the data reported here indicate that the SPI1 activator HilA was not required for the entry of Salmonella
serovar Dublin into A. rhysodes
Prolonged incubation of the bacterial-amoebic cultures resulted in a gradual change in morphology and eventually in the disappearance of the host cells (Fig. ). A related phenomenon has been reported by Ly and Müller (28
) for cocultures of Listeria monocytogenes
species. Listeriae were taken up by the amoebae, and the bacteria replicated intracellularly. However, longer incubations led to release of bacteria and encystment, with the intracellular listeriae losing viability in cysts.
A portion of the observations implied that the loss of salmonellae in A. rhysodes was not simply a reflection of intracellular killing of the bacteria. Salmonella spp., like several other bacterial pathogens, including Mycobacterium, Legionella, and Brucella spp., replicate within membrane-bound vacuoles inside macrophages. While we did not attempt to define the nature of intracellular vesicles, electron microscopy of Salmonella-infected A. rhysodes showed that the bacteria were localized within membrane-bound vacuoles (Fig. ), and some bacteria apparently were replicating (Fig. ). When we used Salmonella serovar Dublin carrying a plasmid with a temperature-sensitive replicon in the intracellular replication experiment, the plasmid segregated as a function of time, implying that a least a portion of the bacteria were actually replicating inside the acanthamoebae (Fig. ).
Finally, since we recovered more bacteria from detached host cells than from cells attached to the culture chamber (Fig. ), it appears that engulfment of Salmonella
serovar Dublin resulted in cytotoxicity, leading to detachment and disintegration of the amoebae, which subsequently resulted in exposure of the bacteria to gentamicin. This resembles the scenario seen with human monocytic cells and MDCK epithelial cells infected with S. enterica
in that the cells are intoxicated by the infecting bacteria (6
SPI1 is responsible for inducing a very rapid apoptotic response in mononuclear phagocytes (31
). Another, later phase of cytotoxicity is dependent on SPI2 and the spv
) and involves apoptosis and interference with the normal dynamics of the actin cytoskeleton. Concomitantly, the loss of infected Acanthamoeba
cells and the apparent growth yields of the bacteria were found to be partly dependent on hilA
and the S. enterica spv-
carrying virulence plasmid (Fig. ). However, it remains to be shown whether the effect of SPI1 and the spv
genes is that of inducing programmed cell death in amoebae, or whether loss of cells is due to other effects of the corresponding virulence proteins.
Free-living amoebae can harbor bacteria inside their cysts, giving them a microhabitat protecting them from environmental hazards. Furthermore, a study by King et al. (26
) showed that the bacterium-protozoan association provides increased numbers of bacteria with increased resistance to free chlorine residuals, which can lead to the persistence of bacteria in chlorine-treated water. It has also been reported that amoeba-grown L. pneumophila
displays increased intracellular survival and replication in macrophages (9
) and that intracellular growth in A. castellanii
affects the monocyte entry mechanism and enhances the virulence of L. pneumophila
Although the present study included only a restricted set of salmonellae and acanthamoebae, and therefore formally may not reflect all possible forms of interactions, our results show that A. rhysodes was able to ingest salmonellae and that subsequent events included intracellular bacterial replication. We also detected a bacterium-mediated cytotoxicity that appeared to be dependent on documented virulence genes, implying that genetic determinants of salmonellae used for invasion and intracellular proliferation in mammals could also be operative in the environment. It thus remains possible that free-living amoebae function as environmental hosts for salmonellae and that such amoebae participate in the transmission of salmonellosis.