spp. are intracellular bacterial pathogens that cause diarrhoeal disease in humans. utilize the host actin cytoskeleton to enter cells, move through the cytoplasm of cells and pass into adjacent cells. Ena/VASP family proteins are highly conserved proteins that participate in actin-dependent dynamic cellular processes. We tested whether Ena/VASP family members VASP (vasodilator-stimulated phosphoprotein), Mena (mammalian-enabled) or EVL (Ena-VASP-like) contribute to spread through cell monolayers. VASP and EVL restricted cell-to-cell spread without significantly altering actin-based motility, whereas Mena had no effect on these processes. Phosphorylation of VASP on Ser153, Ser235 and Thr274 regulated its subcellular distribution and function. VASP derivatives that lack the Ena/VASP homology 1 (EVH1) domain or contain a phosphoablative mutation of Ser153 were defective in restricting spread, indicating that the EVH1 domain and phosphorylation on Ser153 are required for this process. The EVH1 domain and Ser153 of VASP were required for VASP localization to focal adhesions, and localization of VASP to focal adhesions and/or the leading edge was required for restriction of spread. The contribution of the EVH1 domain was from both the donor and the recipient cell, whereas the contribution of Ser153 phosphorylation was only from the donor cell. Thus, unlike host proteins characterized in pathogenesis that promote bacterial spread, VASP and EVL function to limit it. The ability of VASP and EVL to limit spread highlights the critical role of focal adhesion complexes and/or the leading edge in bacterial passage between cells.


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  1. Ally S., Sauer N.J., Loureiro J.J., Snapper S.B., Gertler F.B., Goldberg M.B. (2004). Shigella interactions with the actin cytoskeleton in the absence of Ena/VASP family proteinsCell Microbiol 6355366 [View Article][PubMed]. [Google Scholar]
  2. Applewhite D.A., Barzik M., Kojima S., Svitkina T.M., Gertler F.B., Borisy G.G. (2007). Ena/VASP proteins have an anti-capping independent function in filopodia formationMol Biol Cell 1825792591 [View Article][PubMed]. [Google Scholar]
  3. Auerbuch V., Loureiro J.J., Gertler F.B., Theriot J.A., Portnoy D.A. (2003). Ena/VASP proteins contribute to Listeria monocytogenes pathogenesis by controlling temporal and spatial persistence of bacterial actin-based motilityMol Microbiol 4913611375 [View Article][PubMed]. [Google Scholar]
  4. Barzik M., McClain L.M., Gupton S.L., Gertler F.B. (2014). Ena/VASP regulates mDia2-initiated filopodial length, dynamics, and functionMol Biol Cell 2526042619 [View Article][PubMed]. [Google Scholar]
  5. Bear J.E., Gertler F.B. (2009). Ena/VASP: towards resolving a pointed controversy at the barbed endJ Cell Sci 12219471953 [View Article][PubMed]. [Google Scholar]
  6. Bear J.E., Loureiro J.J., Libova I., Fässler R., Wehland J., Gertler F.B. (2000). Negative regulation of fibroblast motility by Ena/VASP proteinsCell 101717728 [View Article][PubMed]. [Google Scholar]
  7. Benz P.M., Blume C., Seifert S., Wilhelm S., Waschke J., Schuh K., Gertler F., Münzel T., Renné T. (2009). Differential VASP phosphorylation controls remodeling of the actin cytoskeletonJ Cell Sci 12239543965 [View Article][PubMed]. [Google Scholar]
  8. Bilancia C.G., Winkelman J.D., Tsygankov D., Nowotarski S.H., Sees J.A., Comber K., Evans I., Lakhani V., Wood W., other authors. (2014). Enabled negatively regulates diaphanous-driven actin dynamics in vitro and in vivoDev Cell 28394408 [View Article][PubMed]. [Google Scholar]
  9. Blume C., Benz P.M., Walter U., Ha J., Kemp B.E., Renné T. (2007). AMP-activated protein kinase impairs endothelial actin cytoskeleton assembly by phosphorylating vasodilator-stimulated phosphoproteinJ Biol Chem 28246014612 [View Article][PubMed]. [Google Scholar]
  10. Boëda B., Briggs D.C., Higgins T., Garvalov B.K., Fadden A.J., McDonald N.Q., Way M. (2007). Tes, a specific Mena interacting partner, breaks the rules for EVH1 bindingMol Cell 2810711082 [View Article][PubMed]. [Google Scholar]
  11. Breitbach K., Rottner K., Klocke S., Rohde M., Jenzora A., Wehland J., Steinmetz I. (2003). Actin-based motility of Burkholderia pseudomallei involves the Arp 2/3 complex, but not N-WASP and Ena/VASP proteinsCell Microbiol 5385393 [View Article][PubMed]. [Google Scholar]
  12. Breitsprecher D., Kiesewetter A.K., Linkner J., Vinzenz M., Stradal T.E., Small J.V., Curth U., Dickinson R.B., Faix J. (2011). Molecular mechanism of Ena/VASP-mediated actin-filament elongationEMBO J 30456467 [View Article][PubMed]. [Google Scholar]
  13. Butt E., Abel K., Krieger M., Palm D., Hoppe V., Hoppe J., Walter U. (1994). cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) in vitro and in intact human plateletsJ Biol Chem 2691450914517[PubMed]. [Google Scholar]
  14. Chakraborty T., Ebel F., Domann E., Niebuhr K., Gerstel B., Pistor S., Temm-Grove C.J., Jockusch B.M., Reinhard M., other authors. (1995). A focal adhesion factor directly linking intracellularly motile Listeria monocytogenes and Listeria ivanovii to the actin-based cytoskeleton of mammalian cellsEMBO J 1413141321[PubMed]. [Google Scholar]
  15. Chen X.J., Squarr A.J., Stephan R., Chen B., Higgins T.E., Barry D.J., Martin M.C., Rosen M.K., Bogdan S., Way M. (2014). Ena/VASP proteins cooperate with the WAVE complex to regulate the actin cytoskeletonDev Cell 30569584 [View Article][PubMed]. [Google Scholar]
  16. Döppler H.R., Bastea L.I., Lewis-Tuffin L.J., Anastasiadis P.Z., Storz P. (2013). Protein kinase D1-mediated phosphorylations regulate vasodilator-stimulated phosphoprotein (VASP) localization and cell migrationJ Biol Chem 2882438224393 [View Article][PubMed]. [Google Scholar]
  17. Dragoi A.M., Agaisse H. (2014). The serine/threonine kinase STK11 promotes Shigella flexneri dissemination through establishment of cell-cell contacts competent for tyrosine kinase signalingInfect Immun 8244474457 [View Article][PubMed]. [Google Scholar]
  18. Dragoi A.M., Agaisse H. (2015). The class II phosphatidylinositol 3-phosphate kinase PIK3C2A promotes Shigella flexneri dissemination through formation of vacuole-like protrusionsInfect Immun 8316951704 [View Article][PubMed]. [Google Scholar]
  19. Fukumatsu M., Ogawa M., Arakawa S., Suzuki M., Nakayama K., Shimizu S., Kim M., Mimuro H., Sasakawa C. (2012). Shigella targets epithelial tricellular junctions and uses a noncanonical clathrin-dependent endocytic pathway to spread between cellsCell Host Microbe 11325336 [View Article][PubMed]. [Google Scholar]
  20. Gertler F.B., Niebuhr K., Reinhard M., Wehland J., Soriano P. (1996). Mena, a relative of VASP and Drosophila Enabled, is implicated in the control of microfilament dynamicsCell 87227239 [View Article][PubMed]. [Google Scholar]
  21. Goldberg M.B. (2001). Actin-based motility of intracellular microbial pathogensMicrobiol Mol Biol Rev 65595626 [View Article][PubMed]. [Google Scholar]
  22. Gouin E., Gantelet H., Egile C., Lasa I., Ohayon H., Villiers V., Gounon P., Sansonetti P.J., Cossart P. (1999). A comparative study of the actin-based motilities of the pathogenic bacteria Listeria monocytogenes, Shigella flexneri and Rickettsia conoriiJ Cell Sci 11216971708[PubMed]. [Google Scholar]
  23. Gupton S.L., Riquelme D., Hughes-Alford S.K., Tadros J., Rudina S.S., Hynes R.O., Lauffenburger D., Gertler F.B. (2012). Mena binds α5 integrin directly and modulates α5β1 functionJ Cell Biol 198657676 [View Article][PubMed]. [Google Scholar]
  24. Hansen S.D., Mullins R.D. (2010). VASP is a processive actin polymerase that requires monomeric actin for barbed end associationJ Cell Biol 191571584 [View Article][PubMed]. [Google Scholar]
  25. Havrylenko S., Noguera P., Abou-Ghali M., Manzi J., Faqir F., Lamora A., Guérin C., Blanchoin L., Plastino J. (2015). WAVE binds Ena/VASP for enhanced Arp2/3 complex-based actin assemblyMol Biol Cell 265565 [View Article][PubMed]. [Google Scholar]
  26. Heindl J.E., Saran I., Yi C.R., Lesser C.F., Goldberg M.B. (2010). Requirement for formin-induced actin polymerization during spread of Shigella flexneriInfect Immun 78193203 [View Article][PubMed]. [Google Scholar]
  27. Hoffman L.M., Jensen C.C., Kloeker S., Wang C.L., Yoshigi M., Beckerle M.C. (2006). Genetic ablation of zyxin causes Mena/VASP mislocalization, increased motility, and deficits in actin remodelingJ Cell Biol 172771782 [View Article][PubMed]. [Google Scholar]
  28. Ireton K. (2013). Molecular mechanisms of cell-cell spread of intracellular bacterial pathogensOpen Biol 3130079 [View Article][PubMed]. [Google Scholar]
  29. Kapushesky M., Adamusiak T., Burdett T., Culhane A., Farne A., Filippov A., Holloway E., Klebanov A., Kryvych N., other authors. (2012). Gene Expression Atlas update – a value-added database of microarray and sequencing-based functional genomics experimentsNucleic Acids Res 40(D1), D1077D1081 [View Article][PubMed]. [Google Scholar]
  30. Ke Y., Tan Y., Wei N., Yang F., Yang H., Cao S., Wang X., Wang J., Han Y., other authors. (2015). Yersinia protein kinase A phosphorylates vasodilator-stimulated phosphoprotein to modify the host cytoskeletonCell Microbiol 17473485 [View Article][PubMed]. [Google Scholar]
  31. Kespichayawattana W., Rattanachetkul S., Wanun T., Utaisincharoen P., Sirisinha S. (2000). Burkholderia pseudomallei induces cell fusion and actin-associated membrane protrusion: a possible mechanism for cell-to-cell spreadingInfect Immun 6853775384 [View Article][PubMed]. [Google Scholar]
  32. Kim M., Ogawa M., Fujita Y., Yoshikawa Y., Nagai T., Koyama T., Nagai S., Lange A., Fässler R., Sasakawa C. (2009). Bacteria hijack integrin-linked kinase to stabilize focal adhesions and block cell detachmentNature 459578582 [View Article][PubMed]. [Google Scholar]
  33. Knauer O., Binai N.A., Carra G., Beckhaus T., Hanschmann K.M., Renné T., Backert S., Karas M., Wessler S. (2008). Differential phosphoproteome profiling reveals a functional role for VASP in Helicobacter pylori-induced cytoskeleton turnover in gastric epithelial cellsCell Microbiol 1022852296 [View Article][PubMed]. [Google Scholar]
  34. Krause M., Dent E.W., Bear J.E., Loureiro J.J., Gertler F.B. (2003). Ena/VASP proteins: regulators of the actin cytoskeleton and cell migrationAnnu Rev Cell Dev Biol 19541564 [View Article][PubMed]. [Google Scholar]
  35. Krause M., Leslie J.D., Stewart M., Lafuente E.M., Valderrama F., Jagannathan R., Strasser G.A., Rubinson D.A., Liu H., other authors. (2004). Lamellipodin, an Ena/VASP ligand, is implicated in the regulation of lamellipodial dynamicsDev Cell 7571583 [View Article][PubMed]. [Google Scholar]
  36. Kuehl C.J., Dragoi A.M., Agaisse H. (2014). The Shigella flexneri type 3 secretion system is required for tyrosine kinase-dependent protrusion resolution, and vacuole escape during bacterial disseminationPLoS One 9e112738 [View Article][PubMed]. [Google Scholar]
  37. Labrec E.H., Schneider H., Magnani T.J., Formal S.B. (1964). Epithelial cell penetration as an essential step in the pathogenesis of bacillary dysenteryJ Bacteriol 8815031518[PubMed]. [Google Scholar]
  38. Lambrechts A., Kwiatkowski A.V., Lanier L.M., Bear J.E., Vandekerckhove J., Ampe C., Gertler F.B. (2000). cAMP-dependent protein kinase phosphorylation of EVL, a Mena/VASP relative, regulates its interaction with actin and SH3 domainsJ Biol Chem 2753614336151 [View Article][PubMed]. [Google Scholar]
  39. Lindsay S.L., Ramsey S., Aitchison M., Renné T., Evans T.J. (2007). Modulation of lamellipodial structure and dynamics by NO-dependent phosphorylation of VASP Ser239J Cell Sci 12030113021 [View Article][PubMed]. [Google Scholar]
  40. Lo S.H. (2006). Focal adhesions: what's new insideDev Biol 294280291 [View Article][PubMed]. [Google Scholar]
  41. Loureiro J.J., Rubinson D.A., Bear J.E., Baltus G.A., Kwiatkowski A.V., Gertler F.B. (2002). Critical roles of phosphorylation and actin binding motifs, but not the central proline-rich region, for Ena/vasodilator-stimulated phosphoprotein (VASP) function during cell migrationMol Biol Cell 1325332546 [View Article][PubMed]. [Google Scholar]
  42. Mounier J., Laurent V., Hall A., Fort P., Carlier M.F., Sansonetti P.J., Egile C. (1999). Rho family GTPases control entry of Shigella flexneri into epithelial cells but not intracellular motilityJ Cell Sci 11220692080[PubMed]. [Google Scholar]
  43. Niebuhr K., Ebel F., Frank R., Reinhard M., Domann E., Carl U.D., Walter U., Gertler F.B., Wehland J., Chakraborty T. (1997). A novel proline-rich motif present in ActA of Listeria monocytogenes and cytoskeletal proteins is the ligand for the EVH1 domain, a protein module present in the Ena/VASP familyEMBO J 1654335444 [View Article][PubMed]. [Google Scholar]
  44. Plotnikov S.V., Waterman C.M. (2013). Guiding cell migration by tuggingCurr Opin Cell Biol 25619626 [View Article][PubMed]. [Google Scholar]
  45. Sansonetti P.J., Arondel J., Fontaine A., d'Hauteville H., Bernardini M.L. (1991). OmpB (osmo-regulation) and icsA (cell-to-cell spread) mutants of Shigella flexneri: vaccine candidates and probes to study the pathogenesis of shigellosisVaccine 9416422 [View Article][PubMed]. [Google Scholar]
  46. Sansonetti P.J., Mounier J., Prévost M.C., Mège R.M. (1994). Cadherin expression is required for the spread of Shigella flexneri between epithelial cellsCell 76829839 [View Article][PubMed]. [Google Scholar]
  47. Skoble J., Auerbuch V., Goley E.D., Welch M.D., Portnoy D.A. (2001). Pivotal role of VASP in Arp2/3 complex-mediated actin nucleation, actin branch-formation, and Listeria monocytogenes motilityJ Cell Biol 15589100 [View Article][PubMed]. [Google Scholar]
  48. Stamm L.M., Morisaki J.H., Gao L.Y., Jeng R.L., McDonald K.L., Roth R., Takeshita S., Heuser J., Welch M.D., Brown E.J. (2003). Mycobacterium marinum escapes from phagosomes and is propelled by actin-based motilityJ Exp Med 19813611368 [View Article][PubMed]. [Google Scholar]
  49. Teysseire N., Chiche-Portiche C., Raoult D. (1992). Intracellular movements of Rickettsia conorii and R. typhi based on actin polymerizationRes Microbiol 143821829 [View Article][PubMed]. [Google Scholar]
  50. Theriot J.A., Mitchison T.J., Tilney L.G., Portnoy D.A. (1992). The rate of actin-based motility of intracellular Listeria monocytogenes equals the rate of actin polymerizationNature 357257260 [View Article][PubMed]. [Google Scholar]
  51. Tilney M.S., Tilney L.G., Stephens R.E., Merte C., Drenckhahn D., Cotanche D.A., Bretscher A. (1989). Preliminary biochemical characterization of the stereocilia and cuticular plate of hair cells of the chick cochleaJ Cell Biol 10917111723 [View Article][PubMed]. [Google Scholar]
  52. Van Kirk L.S., Hayes S.F., Heinzen R.A. (2000). Ultrastructure of Rickettsia rickettsii actin tails and localization of cytoskeletal proteinsInfect Immun 6847064713 [View Article][PubMed]. [Google Scholar]
  53. Vehlow A., Soong D., Vizcay-Barrena G., Bodo C., Law A.L., Perera U., Krause M. (2013). Endophilin, Lamellipodin, and Mena cooperate to regulate F-actin-dependent EGF-receptor endocytosisEMBO J 3227222734 [View Article][PubMed]. [Google Scholar]
  54. Welch M.D., Iwamatsu A., Mitchison T.J. (1997). Actin polymerization is induced by Arp2/3 protein complex at the surface of Listeria monocytogenesNature 385265269 [View Article][PubMed]. [Google Scholar]
  55. Welch M.D., Rosenblatt J., Skoble J., Portnoy D.A., Mitchison T.J. (1998). Interaction of human Arp2/3 complex and the Listeria monocytogenes ActA protein in actin filament nucleationScience 281105108 [View Article][PubMed]. [Google Scholar]
  56. Zhuang S., Nguyen G.T., Chen Y., Gudi T., Eigenthaler M., Jarchau T., Walter U., Boss G.R., Pilz R.B. (2004). Vasodilator-stimulated phosphoprotein activation of serum-response element-dependent transcription occurs downstream of RhoA and is inhibited by cGMP-dependent protein kinase phosphorylationJ Biol Chem 2791039710407 [View Article][PubMed]. [Google Scholar]
  57. Zimmermann J., Labudde D., Jarchau T., Walter U., Oschkinat H., Ball L.J. (2002). Relaxation, equilibrium oligomerization, and molecular symmetry of the VASP (336-380) EVH2 tetramerBiochemistry 411114311151 [View Article][PubMed]. [Google Scholar]

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