Mutations of arginine residues within the 146-KKRRK-150 motif of the ActA protein of Listeria monocytogenes abolish intracellular motility by interfering with the recruitment of the Arp2/3 complex

The recruitment of actin to the surface of intracellular Listeria monocytog enes and subsequent tail formation is dependent on the expression of the ba cterial surface protein ActA, Of the different functional domains of ActA i dentified thus far, the N-terminal region is absolutely required for actin filament recruitment and intracellular motility, Mutational analysis of thi s domain which abolished actin recruitment by intracellular Listeria monocy togenes identified two arginine residues within the 146-KKRRK-150 motif tha t are essential for its activity. More specifically, recruitment of the Arp 2/3 complex to the bacterial surface, as assessed by immunofluorescence sta ining with antibodies raised against the p21-Arc protein, was not obtained in these mutants. Consistently, treatment of infected cells,vith latrunculi n B, which abrogated actin filament formation, did not affect association o f ActA,vith p21-Arc at the bacterial surface. Thus, the initial recruitment of the Arp2/3 complex to the bacterial surface is independent of, and prec edes, actin polymerisation, Our data suggest that binding of the Arp2/3 com plex is mediated by specific interactions dependent on arginine residues wi thin the 146-KKRRK-150 motif present in ActA.