Salmonella enterica SpvB-mediated ADP-ribosylation as an activator for host cell actin degradation.

The virulence-associated SpvB protein of Salmonella enterica is a mono (ADP-ribosyl)transferase defined to target mammalian actin. Exposure of Acanthamoeba rhysodes cell lysate with SpvB and [32P]nicotinamide adenine dinucleotide (NAD) was here observed to result in labeling of a protein of 43 kDa that subsequently was identified as actin by immunoprecipitation. In parallel, ADP-ribosylation promoted degradation of the protozoan actin. SpvB-mediated actin degradation occurred in the presence of the serine protease inhibitor phenylmethylsulfonylfluoride (PMSF), but was inhibited upon addition of novobiocin, an inhibitor of mono (ADP-ribosyl)transferase activity, or upon addition of EDTA. Infection of A. rhysodes with SpvB-proficient S. enterica serovariant Dublin resulted in cytotoxicity and in characteristic SpvB-mediated actin degradation. Cells infected with SpvB-deficient bacteria showed a decrease in cytotoxicity and lack of actin degradation. Combining these results show that SpvB formally can ADP-ribosylate A. rhysodes actin but that the protozoan cell has the capacity to subsequently degrade ADP-ribosyl-tagged actin. These observations illustrate a hitherto undefined consequence of actin modification, and define a new pathway in the cellular actin dynamics.

Uptake and replication of Salmonella enterica in Acanthamoeba rhysodes.

The ability of salmonellae to become internalized and to survive and replicate in amoebae was evaluated by using three separate serovars of Salmonella enterica and five different isolates of axenic Acanthamoeba spp. In gentamicin protection assays, Salmonella enterica serovar Dublin was internalized more efficiently than Salmonella enterica serovar Enteritidis or Salmonella enterica serovar Typhimurium in all of the amoeba isolates tested. The bacteria appeared to be most efficiently internalized by Acanthamoeba rhysodes. Variations in bacterial growth conditions affected internalization efficiency, but this effect was not altered by inactivation of hilA, a key regulator in the expression of the invasion-associated Salmonella pathogenicity island 1. Microscopy of infected A. rhysodes revealed that S. enterica resided within vacuoles. Prolonged incubation resulted in a loss of intracellular bacteria associated with morphological changes and loss of amoebae. In part, these alterations were associated with hilA and the Salmonella virulence plasmid. The data show that Acanthamoeba spp. can differentiate between different serovars of salmonellae and that internalization is associated with cytotoxic effects mediated by defined Salmonella virulence loci.