THE BACTERIAL ACTIN NUCLEATOR PROTEIN ACTA OF LISTERIA-MONOCYTOGENES CONTAINS MULTIPLE BINDING-SITES FOR HOST MICROFILAMENT PROTEINS

Background: Several intracellular pathogens, including Listeria monocy togenes, use components of the host actin-based cytoskeleton for intra cellular movement and for cell-to-cell spread. These bacterial systems provide relatively simple model systems with which to study actin-bas ed motility. Genetic analysis of L. monocytogenes led to the identific ation of the 90 kD surface-bound ActA polypeptide as the sole bacteria l factor required for the initiation of recruitment of host actin fila ments. Numerous host actin-binding proteins have been localized within the actin-based cytoskeleton that surrounds Listeria once it is insid e a mammalian cell, including alpha-actinin, fimbrin, filamin, villin, ezrin/radixin, profilin and the vasodilator-stimulated phosphoprotein , VASP. Only VASP is known to bind directly to ActA. We sought to dete rmine which regions of the ActA molecule interact with VASP and other components of the host microfilament system. Results: We used the prev iously developed mitochondrial targeting assay to determine regions of the ActA protein that are involved in the recruitment of the host act in-based cytoskeleton. By examining amino-terminally truncated ActA de rivatives for their ability to recruit cytoskeletal proteins, an essen tial element for actin filament nucleation was identified between amin o acids 128 and 151 of ActA. An ActA derivative from which the central proline-rich repeats were deleted retained its ability to recruit fil amentous actin, albeit poorly, but was unable to bind VASP. Conclusion s: Our studies reveal the initial interactions that take place between invading Listeria and host microfilament proteins. The listerial ActA polypeptide contains at least two essential sites that are required f or efficient microfilament assembly: an amino-terminal 23 amino-acid r egion for actin filament nucleation, and VASP-binding proline-rich rep eats. Hence, ActA represents a prototype actin filament nucleator. We suggest that host cell analogues of ActA exist and are important compo nents of structures involved in cell motility.