Abstract
The facultative intracellular human pathogenic bacterium Listeria monocytogenes actively recruits host actin to its surface to achieve motility within infected cells. The bacterial surface protein ActA is solely responsible for this process by mimicking fundamental steps of host cell actin dynamics. ActA, a modular protein, contains an N-terminal actin nucleation site and a central proline-rich motif of the 4-fold repeated consensus sequence FPPPP (FP(4)). This motif is specifically recognized by members of the Ena/VASP protein family. These proteins additionally recruit the profilin-G-actin complex increasing the local concentration of G-actin close to the bacterial surface. By using analytical ultracentrifugation, we show that a single ActA molecule can simultaneously interact with four Ena/VASP homology 1 (EVH1) domains. The four FP(4) sites have roughly equivalent affinities with dissociation constants of about 4 microm. Mutational analysis of the FP(4) motifs indicate that the phenylalanine is mandatory for ActA-EVH1 interaction, whereas in each case exchange of the third proline was tolerated. Finally, by using sedimentation equilibrium centrifugation techniques, we demonstrate that ActA is a monomeric protein. By combining these results, we formulate a stoichiometric model to describe how ActA enables Listeria to utilize efficiently resources of the host cell microfilament for its own intracellular motility.
Highlights
Listeria monocytogenes is a Gram-positive facultative intracellular pathogen causing serious systemic infections in humans with compromised immune systems [1,2,3]
The surface protein ActA from L. monocytogenes efficiently interacts with components of the host cell actin polymerizing machinery [21, 42, 43] presumably by functionally mimicking mammalian proteins such as zyxin and vinculin [21]
Both zyxin and vinculin harbor FP4 motifs similar to those of ActA that are responsible for the recruitment of Ena/vasodilator-stimulated phosphoprotein (VASP) proteins
Summary
Drosophila Enabled; Arp, actinrelated protein; Arc, Arp complex; EVH1, Ena/VASP homology 1; GST, glutathione S-transferase; IRR, inter-repeat region; Mena, mammalian Enabled; PCR, polymerase chain reaction; VASP, vasodilator-stimulated phosphoprotein; BSA, bovine serum albumin; sActA, soluble recombinant ActA. The central proline-rich region of ActA, in contrast to the N-terminal part, is not strictly required for actin assembly and L. monocytogenes motility, its deletion leads to delocalization of VASP and Mena from the bacterial surface, resulting in a drastic reduction in listerial motility and a strongly attenuated virulence [18, 21]. By using sedimentation equilibrium centrifugation techniques, we unambiguously show that ActA is a functional monomer in contradiction to previous reports of ActA dimer formation [33]
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