Knowledge Bank

The actin crosslinking domain (ACD), a product of pathogenic Gram-negative bacteria Vibrio cholera, Vibrio vulnificus and Aeromonas hydrophila, is a potent, actin-specific toxin. Once in the cytoplasm of host cells, ACD catalyzes the intermolecular, amide bond formation between the actin residues glutamic acid-270 and lysine-50 in two different actin monomers. This crosslinking leads to the formation of non-polymerizable actin oligomers and eventual cell rounding. Recently, our laboratory found that actin oligomers potently inhibit formins, a family of actin binding proteins that regulates many actin-dependent processes. This potency of actin oligomers is achieved by binding with high affinity to tandemly organized G-actin-binding domains in formins, an attribute enabled by binding to multiple sites on the formins. Similarly, it is possible that actin oligomers may affect other actin regulatory proteins that have several actin-binding domains organized in close proximity. Ena/VASP is such an actin regulatory protein, which increases the rate of actin polymerization approximately two to three fold. Previously, Ena/VASP had not been identified as a target of bacterial toxins. To study the effects of actin oligomers on Ena-controlled actin polymerization, we employed pyrene polymerization assays. A potent, dose-dependent inhibition of Ena by oligomers was observed both in the presence and absence of profilin, a major regulator of polymerization of actin monomers. We also determined that profilin does not play a significant role in the inhibition mechanism. These results suggest that the mechanism of filament growth inhibition is similar to that of a capping protein where the inhibition occurs once Ena is already associated with the actin filament. The observation of single filament growth in the presence of Ena and oligomers will help to confirm this mechanism. Further understanding of the role of ACD-crosslinked actin oligomers will allow us to create tools to study and control Ena/VASP-mediated processes.