Actin is ADP-ribosylated by the Salmonella enterica virulence-associated protein SpvB

The Salmonella enterica virulence-associated protein SpvB was recently show n to contain a carboxyterminal mono(ADP-ribosyl)transferase domain. We demo nstrate here that the catalytic domain of SpvB as well bacterial extracts c ontaining full-length SpvB modifies a 43 kDa protein from macrophage-like J 774-A.1 and epithelial MUCK cells as shown by label transfer from [P-32]-ni cotinamide adenine dinucleotide (NAD) to the 43 kDa protein. When analysed by two-dimensional gel etectrophoresis, the same protein was modified in ce lls infected with S. enterica serovariant Dublin strain SH9325, whereas inf ection with an isogenic spvB mutant strain did not result in modification. Immunoprecipitation and immunoblotting experiments using SH9325-infected ce lls identified the modified protein as actin. The isolated catalytic domain of SpvB mediated transfer of 32P from [P-32]-NAD to actins from various so urces in vitro, whereas isolated eukaryotic control proteins or bacterial p roteins were not modified, In an in vitro actin polymerization assay, the i solated catalytic SpvB domain prevented the conversion of G actin into F ac tin. Microscopic examination of MDCK cells infected with SH9325 revealed mo rphological changes and loss of filamentous actin content, whereas cells in fected with the spvB mutant remained virtually unaffected. We conclude that actin is a target for an SpvB-mediated modification, most probably ADP-rib osylation, and that the modification of G actin interferes with actin polym erization.