Actin is ADP-ribosylated by the Salmonella enterica virulence-associated protein SpvB.
The Salmonella enterica virulence-associated protein SpvB was recently shown to contain a carboxy-terminal mono(ADP-ribosyl)transferase domain. We demonstrate here that the catalytic domain of SpvB as well bacterial extracts containing full-length SpvB modifies a 43 kDa protein from macrophage-like J774-A.1 and epithelial MDCK cells as shown by label transfer from [32P]-nicotinamide adenine dinucleotide (NAD) to the 43 kDa protein. When analysed by two-dimensional gel electrophoresis, the same protein was modified in cells infected with S. enterica serovariant Dublin strain SH9325, whereas infection with an isogenic spvB mutant strain did not result in modification. Immunoprecipitation and immunoblotting experiments using SH9325-infected cells identified the modified protein as actin. The isolated catalytic domain of SpvB mediated transfer of 32P from [32P]-NAD to actins from various sources in vitro, whereas isolated eukaryotic control proteins or bacterial proteins were not modified. In an in vitro actin polymerization assay, the isolated catalytic SpvB domain prevented the conversion of G actin into F actin. Microscopic examination of MDCK cells infected with SH9325 revealed morphological changes and loss of filamentous actin content, whereas cells infected with the spvB mutant remained virtually unaffected. We conclude that actin is a target for an SpvB-mediated modification, most probably ADP-ribosylation, and that the modification of G actin interferes with actin polymerization.
Tezcan-Merdol
D
D
Microbiology and Tumor Biology Center, Karolinska Institute, Nobels väg 16, 171 77 Stockholm, Sweden.
Nyman
T
T
Lindberg
U
U
Haag
F
F
Koch-Nolte
F
F
Rhen
M
M
D036002:ADP Ribose Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000199:Actins / Q000378:metabolism*; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D002460:Cell Line; D015151:Immunoblotting; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011108:Polymers / Q000378:metabolism; D011233:Precipitin Tests; D019779:Salmonella enterica / Q000201:enzymology* / Q000472:pathogenicity*; D014774:Virulence; D037521:Virulence Factors
D000199:Actins; D011108:Polymers; D037521:Virulence Factors; D000246:Adenosine Diphosphate Ribose; D036002:ADP Ribose Transferases; C415223:spvB protein, Salmonella enterica virulence plasmid; D011065:Poly(ADP-ribose) Polymerases