Three regions within ActA promote Arp2/3 complex-mediated actin nucleationand Listeria monocytogenes motility

The Listeria monocytogenes ActA protein induces actin-based motility by enh ancing the actin nucleating activity of the host Arp2/3 complex. Using syst ematic truncation analysis, we identified a 136-residue NH2-terminal fragme nt that was fully active in stimulating nucleation in vitro. Further deleti on analysis demonstrated that this fragment contains three regions, which a re important for nucleation and share functional and/or limited sequence si milarity with host WASP family proteins: an acidic stretch, an actin monome r-binding region, and a cofilin homology sequence. To determine the contrib ution of each region to actin-based motility, we compared the biochemical a ctivities of ActA derivatives with the phenotypes of corresponding mutant b acteria in cells. The acidic stretch functions to increase the efficiency o f actin nucleation, the rate and frequency of motility, and the effectivene ss of cell-cell spread. The monomer-binding region is required for actin nu cleation in vitro, but not for actin polymerization or motility in infected cells, suggesting that redundant mechanisms may exist to recruit monomer i n host cytosol. The cofilin homology sequence is critical for stimulating a ctin nucleation with the Arp2/3 complex in vitro, and is essential for acti n polymerization and motility in cells. These data demonstrate that each re gion contributes to actin-based motility, and that the cofilin homology seq uence plays a principal role in activation of the Arp2/3 complex, and is an essential determinant of L. monocytogenes pathogenesis.