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* Division of Infectious Diseases, Department of Medicine, and To generate the forces needed for motility,
the plasma membranes of nonmuscle cells adopt an activated state that dynamically reorganizes the actin cytoskeleton. By usurping components from focal contacts and the actin cytoskeleton, the intracellular
pathogens Shigella flexneri and Listeria monocytogenes
use molecular mimicry to create their own actin-based
motors. We raised an antibody (designated FS-1)
against the FEFPPPPTDE sequence of Listeria ActA,
and this antibody: (a) localized at the trailing end of motile intracellular Shigella, (b) inhibited intracellular
locomotion upon microinjection of Shigella-infected
cells, and (c) cross-reacted with the proteolytically derived 90-kD human vinculin head fragment that contains the Vinc-1 oligoproline sequence, PDFPPPPPDL.
Antibody FS-1 reacted only weakly with full-length vinculin, suggesting that the Vinc-1 sequence in full-length
vinculin may be masked by its tail region and that this
sequence is unmasked by proteolysis. Immunofluoresence staining with a monoclonal antibody against the
head region of vinculin (Vin 11-5) localized to the back
of motile bacteria (an identical staining pattern observed with the anti-ActA FS-1 antibody), indicating
that motile bacteria attract a form of vinculin containing an unmasked Vinc-1 oligoproline sequence. Microinjection of submicromolar concentrations of a synthetic Vinc-1 peptide arrested Shigella intracellular
motility, underscoring the functional importance of this
sequence. Western blots revealed that Shigella infection induces vinculin proteolysis in PtK2 cells and generates p90 head fragment over the same 1-3 h time
frame when intracellular bacteria move within the host cell cytoplasm. We also discovered that microinjected
p90, but not full-length vinculin, accelerates rates of
pathogen motility by a factor of 3 ± 0.4 in Shigella-infected PtK2 cells. These experiments suggest that vinculin p90 is a rate-limiting component in actin-based
Shigella motility, and that supplementing cells with p90
stimulates rocket tail growth. Earlier findings demonstrated that vinculin p90 binds to IcsA (Suzuki, T.A., S. Saga, and C. Sasakawa. 1996. J. Biol. Chem. 271:21878-
21885) and to vasodilator-stimulated phosphoprotein
(VASP) (Brindle, N.P.J., M.R. Hold, J.E. Davies, C.J.
Price, and D.R. Critchley. 1996. Biochem. J. 318:753-
757). We now offer a working model in which proteolysis unmasks vinculin's ActA-like oligoproline sequence.
Unmasking of this site serves as a molecular switch that
initiates assembly of an actin-based motility complex
containing VASP and profilin.
Department of Biochemistry and Molecular Biology, University
of Florida College of Medicine, Health Science Center, Gainesville, Florida 32610-0277
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