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The SpvB protein from Salmonella enterica was recently discovered as an actin-ADP-ribosylating toxin. SpvB is most likely delivered via a type-III secretion system into eukaryotic cells and does not have a binding/translocation component. This is in contrast to the family of binary actin-ADP-ribosylating toxins from various Bacillus and Clostridium species. However, there are homologies in amino acid sequences between the C-terminal domain of SpvB and the catalytic domains of the actin-ADP-ribosylating toxins such as C2 toxin from Clostridium botulinum and iota toxin from Clostridium perfringens. We compared the biochemical properties of the catalytic C-terminal domain of SpvB (C/SpvB) with the enzyme components of C2 toxin and iota toxin. The specificity of C/SpvB concerning the modification of G- or F-actin was comparable to the C2 and iota toxins, although there were distinct differences regarding the recognition of actin isoforms. C/SpvB and iota toxin modify both muscle alpha-actin and nonmuscle beta/gamma-actin, whereas C2 toxin only modifies beta/gamma-actin. In contrast to the iota and C2 toxins, C/SpvB possessed no detectable glycohydrolase activity in the absence of a protein substrate. The maximal reaction rates were comparable for all toxins, whereas variable K(m) values for NAD were evident. We identified arginine-177 as the modification site for C/SpvB with the actin homologue protein Act88F from Drosophila.

The virulence factor SpvB is a crucial component for the intracellular growth and infection process of Salmonella enterica. The SpvB protein mediates the ADP-ribosylation of actin in infected cells and is assumed to be delivered directly from the engulfed bacteria into the host cell cytosol. Here we used the binary Clostridium botulinum C2 toxin as a transport system for the catalytic domain of SpvB (C/SpvB) into the host cell cytosol. A recombinant fusion toxin composed of the enzymatically inactive N-terminal domain of C. botulinum C2 toxin (C2IN) and C/SpvB was cloned, expressed, and characterized in vitro and in intact cells. When added together with C2II, the C2IN-C/SpvB fusion toxin was efficiently delivered into the host cell cytosol and ADP-ribosylated actin in various cell lines. The cellular uptake of the fusion toxin requires translocation from acidic endosomes into the cytosol and is facilitated by Hsp90. The N-and C-terminal domains of SpvB are linked by 7 proline residues. To elucidate the function of this proline region, fusion toxins containing none, 5, 7, and 9 proline residues were constructed and analyzed. The existence of the proline residues was essential for the translocation of the fusion toxins into host cell cytosol and thereby determined their cytopathic efficiency. No differences concerning the mode of action of the C2IN-C/SpvB fusion toxin and the C2 toxin were obvious as both toxins induced depolymerization of actin filaments, resulting in cell rounding. The acute cellular responses following ADP-ribosylation of actin did not immediately induce cell death of J774.A1 macrophage-like cells.Several pathogenic bacteria produce toxins and effector proteins, which attack the cytoskeleton of eukaryotic cells by mono-ADP-ribosylation of actin. In past years, we focused on the mode of action of the Clostridium botulinum C2 toxin as the prototype of binary actin-ADP-ribosylating toxins (1). The enzyme component of the C2 toxin (C2I) 3 ADP-ribosylates G-actin at arginine 177 (2). This leads to depolymerization of actin filaments and finally to cell rounding. The proteolytically activated binding/translocation component (C2IIa) forms heptamers, which assemble with C2I and bind to the cellular receptor (3). Following receptor-mediated endocytosis, C2IIa forms pores in the membrane of acidic endosomes. For translocation of the C2I protein through the lumen of these pores, a partial unfolding of C2I is required (4). The subsequent refolding of C2I in the cytosol is facilitated by the host cell chaperone Hsp90 (5). The interaction of C2I with C2IIa is mediated by the N-terminal domain of the C2I protein (C2IN, amino acid residues 1-225). C2IN, which is enzymatically inactive and does not induce cell rounding when applied in combination with C2IIa to cells, was successfully used as an adaptor for the C2IIa-mediated delivery of different proteins into the cytosol of eukaryotic cells (6).The SpvB protein from Salmonella enterica was identified as a new member of bacterial actin-ADP-ribosylating enzymes...