Modification of actin by bacterial protein toxins and effectors

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Various bacterial protein toxins and effectors directly modify actin of eukaryotic target cells. Best known is the modification of G-actin by members of the family of binary ADP-ribosylating protein toxins. The prototype of this toxin family is Clostridium botulinum C2 toxin. These toxins ADP-ribosylate G-actin at Arg177. The modification (i) inhibits actin polymerization and (ii) turns actin into a capping protein, which inhibits the polymerization of unmodified actin. Recently, it was reported that the SpvB protein from Salmonella enterica causes redistribution of the cytoskeleton of target cells, and possesses ADP-ribosyltransferase activity that results in modification of actin. In contrast to binary actin-ADP-ribosylating toxins, the SpvB protein is directly delivered from the pathogen into host cells via type-III-secretion. The main objective of this proposal is to elucidate at the molecular and cellular level the cytotoxic effects and mode of action of SpvB that results in the rearrangement of the actin cytoskeleton. To achieve this aim, we propose 1) to characterize in detail the SpvB-induced ADP-ribosylation of actin, including regulation of the ADP-ribosyltransferase activity of the of SpvB by cytosolic factors, 2) to identify the modification of actin by SpvB, and 3) to determine the functional consequences of the SpvB-mediated actin modification on the kinetics of actin nucleation, polymerization, and interaction with various actin-binding proteins, using biochemical methods and imaging techniques and fluorescence microcopy in intact cells.