Actin and Phosphoinositide Binding by the ActA Protein of the Bacterial Pathogen Listeria monocytogenes

Cicchetti, Gregor; Maurer, Patrik; Wagener, Patricia; Kocks, Christine

doi:10.1074/jbc.274.47.33616

Cited by 42 publications

(72 citation statements)

References 55 publications

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“…These results confirm the abnormally low sedimentation velocity of 2.6 S and low diffusion coefficient observed in analytical ultracentrifugation experiments which indicate an elongated monomeric ActA molecule, in correspondence to recently published data (42). DISCUSSION 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 FP 4 motifs similar to those of ActA that are responsible for the recruitment of Ena/VASP proteins.…”

Section: Acta Contains Four Equivalent Moderately Strong Binding Sitsupporting

confidence: 80%

ActA from Listeria monocytogenes Can Interact with Up to Four Ena/VASP Homology 1 Domains Simultaneously

Machner¹,

Urbanke²,

Barzik³

et al. 2001

Journal of Biological Chemistry

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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 M. 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.

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Section: Acta Contains Four Equivalent Moderately Strong Binding Sitsupporting

confidence: 80%

ActA from Listeria monocytogenes Can Interact with Up to Four Ena/VASP Homology 1 Domains Simultaneously

Machner¹,

Urbanke²,

Barzik³

et al. 2001

Journal of Biological Chemistry

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“…A similar effect is seen using ActA lacking the entire aminoterminal region (amino acids 31 to 262) (159). An ActA fragment that consists of amino acids 60 to 100 binds actin, as measured in the same pyrene-actin assay, and a glutathione S-transferase (GST) fusion to ActA amino acids 62 to 103 binds fluorescently labeled monomeric actin (28,195). Mutagenesis of charged residues to alanine within amino acids 60 to 66, 70 to 77, or 80 to 88 decreases the binding of the mutant ActA to monomeric actin (86a), indicating that the entire region between amino acids 60 and 88 is involved in binding to monomeric actin (86a).…”

Section: Regions Of Acta That Bind Monomeric Actinmentioning

confidence: 66%

“…ActA amino acids 30 to 263 fused to a linker and expressed on the bacterial surface mediate actin tail assembly in cytoplasmic extracts (86), which demonstrates that the region is sufficient for actin tail assembly. When ActA is purified and bound to beads, the same region of ActA polymerizes actin in cytoplasmic extracts (28).…”

Section: Regions Of Acta That Bind Monomeric Actinmentioning

confidence: 99%

Actin-Based Motility of Intracellular Microbial Pathogens

Goldberg

2001

Microbiol Mol Biol Rev

199

188

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A diverse group of intracellular microorganisms, including Listeria monocytogenes, Shigella spp., Rickettsia spp., and vaccinia virus, utilize actin-based motility to move within and spread between mammalian host cells. These organisms have in common a pathogenic life cycle that involves a stage within the cytoplasm of mammalian host cells. Within the cytoplasm of host cells, these organisms activate components of the cellular actin assembly machinery to induce the formation of actin tails on the microbial surface. The assembly of these actin tails provides force that propels the organisms through the cell cytoplasm to the cell periphery or into adjacent cells. Each of these organisms utilizes preexisting mammalian pathways of actin rearrangement to induce its own actin-based motility. Particularly remarkable is that while all of these microbes use the same or overlapping pathways, each intercepts the pathway at a different step. In addition, the microbial molecules involved are each distinctly different from the others. Taken together, these observations suggest that each of these microbes separately and convergently evolved a mechanism to utilize the cellular actin assembly machinery. The current understanding of the molecular mechanisms of microbial actin-based motility is the subject of this review

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“…The finding of a phosphoinositide binding site at amino acids 184 -202 on ActA suggested that this binding site could account for the ability of the bacterium to attract these phosphoinositides (9). It was proposed that the binding of PtdIns(4,5)P 2 by ActA might serve to deliver this phosphoinositide to the actin polymerization zone of Listeria and uncap actin filaments.…”

Section: Effects Of Reducing Phosphoinositide Levels On Listeria Intrmentioning

confidence: 99%

“…The bacterial surface protein ActA directly stimulates actin assembly, bypassing the receptor-mediated signal transduction pathways that induce actin filament formation during phagocytosis and chemotaxis (8). Curiously, ActA has a phosphoinositide binding site in the amino-terminal region that is located within amino acids 184 -202 (9,10), and binding of PtdIns(4,5)P 2 and PtdIns(3,4,5)P 3 causes a conformational change in the amino terminus of ActA. However, in vivo phosphoinositide localization studies have not been performed, and the physiological significance of the inositide binding site has not been examined.…”

mentioning

confidence: 99%

Phosphoinositide 3-Kinase Is Required for Intracellular Listeria monocytogenes Actin-based Motility and Filopod Formation

Sidhu

Laryngakis

et al. 2005

Journal of Biological Chemistry

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Motile nonmuscle cells concentrate phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P 3 ) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P 2 ) in areas of new actin filament assembly. There is great interest in assessing the in vivo functional significance of these phosphoinositides, and we have used Listeria monocytogenes to explore the contribution of PtdIns(3,4,5)P 3 and PtdIns(4,5)P 2 to its actin-based motility. In Listeria-infected PtK2 cells Aktpleckstrin homology (PH)-green fluorescent protein (GFP) and phospholipase C␦ (PLC␦)-PH-GFP both first concentrate at the front of motile Listeria, subsequently surrounding the bacterium and then concentrating in the actin filament tail. Surprisingly, Listeria ActA mutant strains lacking the putative phosphoinositide binding site are also able to concentrate these probes. Reduction of available PtdIns(3,4,5)P 3 by expression of Akt-PH-GFP and available PtdIns(4,5)P 2 by expression of PLC␦-PH-GFP both significantly slow Listeria actin-based movement. Treatment of cells with the PI 3-kinase inhibitor, LY294002, dissociates Akt-PH but not PLC␦-PH, from the bacterial surface and cell membranes, and results in near complete inhibition of Listeria actin-based motility and filopod formation. Removal of LY294002 results in rapid and full recovery of Akt-PH localization, Listeria actin-based motility, and filopod formation. These findings suggest that PtdIns(4,5)P 2 is concentrated at the surface of Listeria and serves as the substrate for PtdIns(3,4,5)P 3 production, indicating a central role for PI 3-kinases in Listeria intracellular actinbased motility and filopod formation.The onset of actin-based cell motility in nonmuscle cells is signaled by the increased turnover of phosphoinositides, and phosphoinositides are known to interact with and regulate multiple actin-binding proteins, kinases, and guanine nucleotide exchange factors (1, 2). Following exposure to various agonists that stimulate cell migration, plasma membrane PtdIns(4,5)P 2 1 is converted to PtdIns(3,4,5)P 3 , which is then concentrated at the leading edge of the cell membrane. The accumulation of PtdIns(3,4,5)P 3 at these sites is associated with the formation of new actin filaments (3). The mechanisms by which these lipid products induce actin assembly are under active investigation and include the uncapping of actin filaments and stimulation of actin nucleation (2). The shape changes associated with chemotaxis, cell spreading, and phagocytosis are complex and make analysis of rates and directionality of actin assembly difficult. Furthermore, these events involve several steps of receptor-mediated signal transduction. Listeria intracellular infection and movement is a particularly useful model for examining the parameters of in vivo actin assembly, allowing discrete temporal and spatial resolution of actin filament formation. Intracellular Listeria infection of tissue culture cells is accomplished by simply overlaying the bacteria on the surface of host cells. Internalins found on the surface...

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Actin and Phosphoinositide Binding by the ActA Protein of the Bacterial Pathogen Listeria monocytogenes

Cited by 42 publications

References 55 publications

ActA from Listeria monocytogenes Can Interact with Up to Four Ena/VASP Homology 1 Domains Simultaneously

ActA from Listeria monocytogenes Can Interact with Up to Four Ena/VASP Homology 1 Domains Simultaneously

Actin-Based Motility of Intracellular Microbial Pathogens

Phosphoinositide 3-Kinase Is Required for Intracellular Listeria monocytogenes Actin-based Motility and Filopod Formation

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