The bacterial actin nucleator protein ActA of Listeria monocytogenes contains multiple binding sites for host microfilament proteins.
BACKGROUND
Several intracellular pathogens, including Listeria monocytogenes, use components of the host actin-based cytoskeleton for intracellular movement and for cell-to-cell spread. These bacterial systems provide relatively simple model systems with which to study actin-based motility. Genetic analysis of L. monocytogenes led to the identification of the 90 kD surface-bound ActA polypeptide as the sole bacterial factor required for the initiation of recruitment of host actin filaments. Numerous host actin-binding proteins have been localized within the actin-based cytoskeleton that surrounds Listeria once it is inside 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 determine which regions of the ActA molecule interact with VASP and other components of the host microfilament system.


RESULTS
We used the previously developed mitochondrial targeting assay to determine regions of the ActA protein that are involved in the recruitment of the host actin-based cytoskeleton. By examining amino-terminally truncated ActA derivatives for their ability to recruit cytoskeletal proteins, an essential element for actin filament nucleation was identified between amino acids 128 and 151 of ActA. An ActA derivative from which the central proline-rich repeats were deleted retained its ability to recruit filamentous actin, albeit poorly, but was unable to bind VASP.


CONCLUSIONS
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 for efficient microfilament assembly: an amino-terminal 23 amino-acid region for actin filament nucleation, and VASP-binding proline-rich repeats. Hence, ActA represents a prototype actin filament nucleator. We suggest that host cell analogues of ActA exist and are important components of structures involved in cell motility.
Pistor
S
S
Gesellschaft für Biotechnologische Forschung, Abteilung Zellbiologie und Immunologie, Braunschweig, Germany.
Chakraborty
T
T
Walter
U
U
Wehland
J
J
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D015815:Cell Adhesion Molecules / Q000378:metabolism; D002460:Cell Line; D003598:Cytoskeletal Proteins / Q000378:metabolism; D008089:Listeria monocytogenes / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008840:Microfilament Proteins / Q000378:metabolism*; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D015388:Organelles / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D011392:Proline / Q000378:metabolism; D011817:Rabbits; D014162:Transfection; D000098662:Vasodilator-Stimulated Phosphoprotein
D000199:Actins; D001426:Bacterial Proteins; D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D008840:Microfilament Proteins; D010750:Phosphoproteins; D000098662:Vasodilator-Stimulated Phosphoprotein; C072980:actA protein, Listeria monocytogenes; D011392:Proline