INTRODUCTION Listeria monocytogenes is a gram positive facultative intracellular pathogen for humans and animals with a significant mortality rate in unborn children and elderly or immunocompromised individuals (Gellin and Broome, 1989). Of the listerial virulence factors, the bacterial surface protein ActA is responsible for the actin-based intra- and intercellular motility of this pathogen in the cytosol of infected host cells. Isogenic mutants of L. monocytogenes lacking the surface- bound ActA polypeptide no longer interact with cytoskeletal elements and are non-motile. As a consequence, these mutants are attenuated in a mouse infection model (Domann et al., 1992; Kocks et al., 1992). Using an organelle targeting transfection system, where ActA is directed to the mitochondrial surface, we were able to ascribe specific functions to discrete domains of the ActA protein (Pistor et al., 1994). These studies revealed that at least two functional domains were important for the interaction of ActA with the host cell cytoskeleton. The first region comprising the centrally located proline-rich repeat region is required for binding to the focal contact proteins VASP and Mena (Pistor et al., 1995; Chakraborty et al., 1995; Gertler et al., 1996). Nevertheless, Listeria mutants harbouring a deletion of this region of ActA are still capable of actin recruitment to the surface of mutant bacteria, albeit to a lesser extent, suggesting that the VASP/Mena proteins are dispensable for initiation of actin assembly on the bacterial surface (Lasa et al., 1995; Smith et al., 1996; Niebuhr et al., 1997). On the other hand, the N-terminal domain of ActA was found to be absolutely essential for actin recruitment. We and others have demonstrated that the first 264 amino acids of the amino terminus of ActA contain all the information necessary to initiate actin filament assembly and to generate tail formation. Additionally, transfection of eukaryotic cells with several N- terminally truncated actA derivatives allowed us to define a region rich in positively charged amino acid residues (amino acid 129-153) as essential for actin assembly (Pistor et al., 1995). Similar results were obtained by Friederich et al. (1995) by fusing actA sequences to the CAAX-box that targeted ActA to the cellular membrane of transfected cells. These results were further corroborated by studying intracellular motility of mutants lacking either of these regions (Lasa et al., 1995; Smith et al., 1996). 3277 Journal of Cell Science 113, 3277-3287 (2000) Printed in Great Britain © The Company of Biologists Limited 2000 JCS1299 The recruitment of actin to the surface of intracellular Listeria monocytogenes and subsequent tail formation is dependent on the expression of the bacterial surface protein ActA. Of the different functional domains of ActA identified thus far, the N-terminal region is absolutely required for actin filament recruitment and intracellular motility. Mutational analysis of this domain which abolished actin recruitment by intracellular Listeria monocytogenes identified two arginine residues within the 146-KKRRK-150 motif that are essential for its activity. More specifically, recruitment of the Arp2/3 complex to the bacterial surface, as assessed by immunofluorescence staining with antibodies raised against the p21-Arc protein, was not obtained in these mutants. Consistently, treatment of infected cells with latrunculin B, which abrogated actin filament formation, did not affect association of ActA with p21-Arc at the bacterial surface. Thus, the initial recruitment of the Arp2/3 complex to the bacterial surface is independent of, and precedes, actin polymerisation. Our data suggest that binding of the Arp2/3 complex is mediated by specific interactions dependent on arginine residues within the 146-KKRRK-150 motif present in ActA. Key words: Actin nucleation, Scar, WASP, ActA, Listeria monocytogenes SUMMARY 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 Susanne Pistor 1, *, Lothar Gröbe 1 , Antonio S. Sechi 1 , Eugen Domann 2 , Birgit Gerstel 1 , Laura M. Machesky 3 , Trinad Chakraborty 2 and Jürgen Wehland 1 1 Department of Cell Biology, Gesellschaft für Biotechnologische Forschung, Mascheroder Weg 1, D-38124 Braunschweig, Germany 2 Institut für Medizinische Mikrobiologie, Justus-Liebig Universität Giessen, D-35392 Giessen, Germany 3 Department of Biochemistry, University of Birmingham, Edgbaston, Birmingham, B15-2TT, UK *Author for correspondence (e-mail: spi@biobase.de) Accepted 3 July 2000; published on WWW 22 August