Shigella, the causative agent of bacillary dysentery, invades epithelial cells in a process involving Src tyrosine kinase signaling. Cortactin, a ubiquitous actin-binding protein present in structures of dynamic actin assembly, is the major protein tyrosine phosphorylated during Shigella invasion. Here, we report that RNA interference silencing of cortactin expression, as does Src inhibition in cells expressing kinase-inactive Src, interferes with actin polymerization required for the formation of cellular extensions engulfing the bacteria. Shigella invasion induced the recruitment of cortactin at plasma membranes in a tyrosine phosphorylation–dependent manner. Overexpression of wild-type forms of cortactin or the adaptor protein Crk favored Shigella uptake, and Arp2/3 binding–deficient cortactin derivatives or an Src homology 2 domain Crk mutant interfered with bacterial-induced actin foci formation. Crk was shown to directly interact with tyrosine-phosphorylated cortactin and to condition cortactin-dependent actin polymerization required for Shigella uptake. These results point at a major role for a Crk–cortactin complex in actin polymerization downstream of tyrosine kinase signaling.

Introduction

Shigella flexneri, a gram-negative bacterium, causes bacillary dysentery in humans by invading the colonic epithelium and eliciting an intense inflammatory reaction that leads to destruction of this epithelium (Labrec et al., 1964; Sansonetti, 1998). Bacterial colonization of the mucosa depends on its ability to invade epithelial cells and to spread from cell to cell (Suzuki and Sasakawa, 2001). The ability of enteroinvasive pathogens such as Shigella to induce its internalization into cells that are normally nonphagocytic is a key event to its virulence. The “zipper”-like process used by Yersinia and Listeria is based on a high affinity interaction between a bacterial surface ligand and a host cell surface receptor, and implicates moderate cytoskeletal rearrangements (Gruenheid and Finlay, 2003). In contrast, the “triggering” process used by Salmonella and Shigella involves several bacterial factors and important rearrangements of the actin cytoskeleton leading to the formation of cell extensions that rise several micrometers above the cell surface and engulf the bacterium in a large vacuole (Galan, 2001). During the triggering process, a specialized type III secretion system determines the integration bacterial components into host cell membranes to form a so-called “translocon,” which allows the subsequent delivery of type III effectors into the cell cytosol (Hueck, 1998). In the case of Salmonella, cytoskeleton reorganization is mediated by translocated type III effectors, the SopE and SptP proteins that act as a guanosine exchange factor and GTPase-activating protein toward the Rho GTPases Cdc42 and Rac, respectively (Galan, 2001). Although phenotypically similar, Shigella invasion implicates a different mechanism, as IpaC, a component of the translocon, triggers signals that lead to actin polymerization (Tran Van Nhieu et al., 1999). During the initial phase of Shigella entry, actin polymerization depends on Rho GTPases and Src tyrosine kinase activation, but the molecular links between these two pathways have not been identified (Duménil et al., 1998; Mounier et al., 1999). The Abl tyrosine kinase family was recently shown to be required for Shigella invasion and may provide such a link because Abl kinases are activated by Src and have been involved in F-actin dynamics (Plattner et al., 1999; Woodring et al., 2003). During Shigella invasion, Abl may activate Rho GTPases through phosphorylation of the Crk adaptor protein (Burton et al., 2003).

Another candidate that could link Rho GTPases and Src pathways is cortactin, an actin-binding protein recruited in Shigella entry foci, which is tyrosine phosphorylated in an Src kinase–dependent manner during Shigella entry (Wu et al., 1991; Dehio et al., 1995; Duménil et al., 1998). Cortactin distributes to sites of dynamic actin assembly, including lamellipodia, podosomes, and invadopodia (Wu and Parsons, 1993; Bowden et al., 1999).

Cortactin possesses a unique multidomain structure consisting of an acidic domain at the amino terminus (NTA) followed by 6.5 tandem repeats containing the F-actin binding site, a proline-rich region containing the three tyrosine residues (421, 466, and 482) that are phosphorylated by Src-related kinases (Huang et al., 1998), and a carboxy-terminal Src homology (SH) 3 domain. Cortactin binds to the Arp2/3 complex and stimulates its actin nucleation activity in vitro (Mullins et al., 1998; Weed et al., 2000; Uruno et al., 2001). Stimulation of cortactin-mediated actin nucleation is enhanced by its binding to F-actin (Uruno et al., 2001; Weaver et al., 2001) and to WIP, a protein involved in filopodia formation (Kinley et al., 2003). Binding to the Arp2/3 complex occurs through the cortactin tripeptide motif “DDW” located in its amino-terminal NTA domain, a motif that is conserved in many Arp2/3-binding/activating proteins such as HS1, Abp1, and WASP family proteins (Olazabal and Machesky, 2001). Cortactin-dependent actin nucleation may be relevant for the genesis of filopodial extensions such as dendritic spine (Hering and Sheng, 2003). Cortactin was also reported to prevent the debranching of Arp2/3–F-actin networks (Weaver et al., 2001).

Results

Cortactin is required for actin polymerization at the Shigella entry site and for efficient bacterial uptake

To inhibit cortactin expression, HeLa cells were transfected with an siRNA duplex corresponding to the 5′ sequence of the cortactin mRNA (dsRNA1). Immunofluorescence staining of transfected cells indicated that inhibition of cortactin expression was maximal 24 h after transfection of dsRNA1, with 74% of cells showing no detectable cortactin. Consistently, anti-cortactin Western blot analysis indicated a 70% decrease in cortactin levels in cells transfected with dsRNA1, compared with control samples (Fig. 1 

a, cortactin), whereas no difference in the levels of expression of actin or Arp3 could be detected (Fig. 1 a, arp3 and actin). F-actin staining indicated that cells transfected with dsRNA1 showed little modification of their actin cytoskeleton (unpublished data). The effects of cortactin inhibition on Shigella uptake were analyzed by infecting dsRNA1-treated cells with Shigella. As shown in Fig. 1 b, bacterial uptake in cells treated with dsRNA1 was decreased by fivefold compared with control cells. This decrease was still observed even after 40 min of bacterial challenge, showing that the invasion process was strongly inhibited in dsRNA1-treated cells and not simply delayed (Fig. 1 b). In dsRNA1-transfected cells, only weak actin polymerization was detected at the site of bacterial contact (Fig. 1 c), whereas as expected, massive actin polymerization was observed at entry sites in untransfected cells (unpublished data) or in scrambled dsRNA1-transfected cells (Fig. 1 c). The number of entry structures showing actin-rich extensions was decreased 10 times in dsRNA1-treated cells compared with control cells, as 90% of Shigella-induced structures in dsRNA1-treated cells appeared abortive (Fig. 1, c and d).

Conversely, overexpression of Flag-tagged full-length (FL) cortactin led to a twofold increase in bacterial uptake compared with mock cells (Fig. S1 and Fig. 2 

a;

Acknowledgments

We thank Javier Pizarro-Cerda for technical help with sucrose gradients. We thank Thomas Meyer for helpful suggestions with siRNA experiments.

L. Bougnères received a fellowship from the French Ministry of Higher Education and Research and from the Fondation pour la Recherche Médicale. P.J. Sansonetti is a Howard Hughes Medical Institute Scholar.

References

References
Abassi, Y.A., and K. Vuori.
2002
. Tyrosine 221 in Crk regulates adhesion-dependent membrane localization of Crk and Rac and activation of Rac signaling.
EMBO J.
21
:
4571
–4582.
Allaoui, A., P.J. Sansonetti, and C. Parsot.
1992
. MxiJ, a lipoprotein involved in secretion of Shigella Ipa invasins, is homologous to YscJ, a secretion factor of the Yersinia Yop proteins.
J. Bacteriol.
174
:
7661
–7669.
Bowden, E.T., M. Barth, D. Thomas, R.I. Glazer, and S.C. Mueller.
1999
. An invasion-related complex of cortactin, paxillin and PKCμ associates with invadopodia at sites of extracellular matrix degradation.
Oncogene.
18
:
4440
–4449.
Burton, E.A., R. Plattner, and A.M. Pendergast.
2003
. Abl tyrosine kinases are required for infection by Shigella flexneri.
EMBO J.
22
:
5471
–5479.
Cheresh, D.A., J. Leng, and R.L. Klemke.
1999
. Regulation of cell contraction and membrane ruffling by distinct signals in migratory cells.
J. Cell Biol.
146
:
1107
–1116.
Dehio, C., M.C. Prevost, and P.J. Sansonetti.
1995
. Invasion of epithelial cells by Shigella flexneri induces tyrosine phosphorylation of cortactin by a pp60c-src-mediated signaling pathway.
EMBO J.
14
:
2471
–2482.
Duménil, G., J.C. Olivo, S. Pellegrini, M. Fellous, P.J. Sansonetti, and G.T. Nhieu.
1998
. Interferon α inhibits a Src-mediated pathway necessary for Shigella-induced cytoskeletal rearrangements in epithelial cells.
J. Cell Biol.
143
:
1003
–1012.
Galan, J.E.
2001
. Salmonella interactions with host cells: type III secretion at work.
Annu. Rev. Cell Dev. Biol.
17
:
53
–86.
Garcia, M.I., P. Gounon, P. Courcoux, A. Labigne, and C. Le Bouguenec.
1996
. The afimbrial adhesive sheath encoded by the afa-3 gene cluster of pathogenic Escherichia coli is composed of two adhesins.
Mol. Microbiol.
19
:
683
–693.
Gruenheid, S., and B.B. Finlay.
2003
. Microbial pathogenesis and cytoskeletal function.
Nature.
422
:
775
–781.
Head, J.A., D. Jiang, M. Li, L.J. Zorn, E.M. Schaefer, J.T. Parsons, and S.A. Weed.
2003
. Cortactin tyrosine phosphorylation requires Rac1 activity and association with the cortical actin cytoskeleton.
Mol. Biol. Cell.
14
:
3216
–3229.
Hering, H., and M. Sheng.
2003
. Activity-dependent redistribution and essential role of cortactin in dendritic spine morphogenesis.
J. Neurosci.
23
:
11759
–11769.
Huang, C., J. Liu, C.C. Haudenschild, and X. Zhan.
1998
. The role of tyrosine phosphorylation of cortactin in the locomotion of endothelial cells.
J. Biol. Chem.
273
:
25770
–25776.
Hueck, C.J.
1998
. Type III protein secretion systems in bacterial pathogens of animals and plants.
Microbiol. Mol. Biol. Rev.
62
:
379
–433.
Kinley, A.W., S.A. Weed, A.M. Weaver, A.V. Karginov, E. Bissonette, J.A. Cooper, and J.T. Parsons.
2003
. Cortactin interacts with WIP in regulating Arp2/3 activation and membrane protrusion.
Curr. Biol.
13
:
384
–393.
Klemke, R.L., J. Leng, R. Molander, P.C. Brooks, K. Vuori, and D.A. Cheresh.
1998
. CAS/Crk coupling serves as a “molecular switch” for induction of cell migration.
J. Cell Biol.
140
:
961
–972.
Labrec, E.H., H. Schneider, T.J. Magnani, and S.B. Formal.
1964
. Epithelial cell penetration as an essential step in the pathogenesis of bacillary dysentery.
J. Bacteriol.
88
:
1503
–1518.
Lamorte, L., S. Rodrigues, V. Sangwan, C.E. Turner, and M. Park.
2003
. Crk associates with a multimolecular Paxillin/GIT2/β-PIX complex and promotes Rac-dependent relocalization of Paxillin to focal contacts.
Mol. Biol. Cell.
14
:
2818
–2831.
Matsuda, M., S. Tanaka, S. Nagata, A. Kojima, T. Kurata, and M. Shibuya.
1992
. Two species of human CRK cDNA encode proteins with distinct biological activities.
Mol. Cell. Biol.
12
:
3482
–3489.
Miki, H., H. Yamaguchi, S. Suetsugu, and T. Takenawa.
2000
. IRSp53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling.
Nature.
408
:
732
–735.
Mounier, J., F.K. Bahrani, and P.J. Sansonetti.
1997
. Secretion of Shigella flexneri Ipa invasins on contact with epithelial cells and subsequent entry of the bacterium into cells are growth stage dependent.
Infect. Immun.
65
:
774
–782.
Mounier, J., V. Laurent, A. Hall, P. Fort, M.F. Carlier, P.J. Sansonetti, and C. Egile.
1999
. Rho family GTPases control entry of Shigella flexneri into epithelial cells but not intracellular motility.
J. Cell Sci.
112
:
2069
–2080.
Mullins, R.D., J.A. Heuser, and T.D. Pollard.
1998
. The interaction of Arp2/3 complex with actin: nucleation, high affinity pointed end capping, and formation of branching networks of filaments.
Proc. Natl. Acad. Sci. USA.
95
:
6181
–6186.
Nagashima, K., A. Endo, H. Ogita, A. Kawana, A. Yamagishi, A. Kitabatake, M. Matsuda, and N. Mochizuki.
2002
. Adaptor protein Crk is required for ephrin-B1-induced membrane ruffling and focal complex assembly of human aortic endothelial cells.
Mol. Biol. Cell.
13
:
4231
–4242.
Okada, S., and J.E. Pessin.
1997
. Insulin and epidermal growth factor stimulate a conformational change in Rap1 and dissociation of the CrkII-C3G complex.
J. Biol. Chem.
272
:
28179
–28182.
Okamura, H., and M.D. Resh.
1995
. p80/85 cortactin associates with the Src SH2 domain and colocalizes with v-Src in transformed cells.
J. Biol. Chem.
270
:
26613
–26618.
Olazabal, I.M., and L.M. Machesky.
2001
. Abp1p and cortactin, new “hand-holds” for actin.
J. Cell Biol.
154
:
679
–682.
Plattner, R., L. Kadlec, K.A. DeMali, A. Kazlauskas, and A.M. Pendergast.
1999
. c-Abl is activated by growth factors and Src family kinases and has a role in the cellular response to PDGF.
Genes Dev.
13
:
2400
–2411.
Sansonetti, P.J.
1998
. Pathogenesis of shigellosis: from molecular and cellular biology of epithelial cell invasion to tissue inflammation and vaccine development.
Jpn. J. Med. Sci. Biol.
51
(Suppl):
S69
–S80.
Sansonetti, P.J., D.J. Kopecko, and S.B. Formal.
1982
. Involvement of a plasmid in the invasive ability of Shigella flexneri.
Infect. Immun.
35
:
852
–860.
Suzuki, T., and C. Sasakawa.
2001
. Molecular basis of the intracellular spreading of Shigella.
Infect. Immun.
69
:
5959
–5966.
Takino, T., M. Tamura, H. Miyamori, M. Araki, K. Matsumoto, H. Sato, and K.M. Yamada.
2003
. Tyrosine phosphorylation of the CrkII adaptor protein modulates cell migration.
J. Cell Sci.
116
:
3145
–3155.
Tran Van Nhieu, G., and R.R. Isberg.
1991
. The Yersinia pseudotuberculosis invasin protein and human fibronectin bind to mutually exclusive sites on the α5β1 integrin receptor.
J. Biol. Chem.
266
:
24367
–24375.
Tran Van Nhieu, G., E. Caron, A. Hall, and P.J. Sansonetti.
1999
. IpaC induces actin polymerization and filopodia formation during Shigella entry into epithelial cells.
EMBO J.
18
:
3249
–3262.
Turner, C.E.
2000
. Paxillin and focal adhesion signaling.
Nat. Cell Biol.
2
:
E231
–E236.
Uruno, T., J. Liu, P. Zhang, Y. Fan, C. Egile, R. Li, S.C. Mueller, and X. Zhan.
2001
. Activation of Arp2/3 complex-mediated actin polymerization by cortactin.
Nat. Cell Biol.
3
:
259
–266.
Uruno, T., J. Liu, Y. Li, N. Smith, and X. Zhan.
2003
. Sequential interaction of actin-related proteins 2 and 3 (Arp2/3) complex with neural Wiscott-Aldrich syndrome protein (N-WASP) and cortactin during branched actin filament network formation.
J. Biol. Chem.
278
:
26086
–26093.
Weaver, A.M., A.V. Karginov, A.W. Kinley, S.A. Weed, Y. Li, J.T. Parsons, and J.A. Cooper.
2001
. Cortactin promotes and stabilizes Arp2/3-induced actin filament network formation.
Curr. Biol.
11
:
370
–374.
Weaver, A.M., J.E. Heuser, A.V. Karginov, W.L. Lee, J.T. Parsons, and J.A. Cooper.
2002
. Interaction of cortactin and N-WASp with Arp2/3 complex.
Curr. Biol.
12
:
1270
–1278.
Weaver, A.M., M.E. Young, W.L. Lee, and J.A. Cooper.
2003
. Integration of signals to the Arp2/3 complex.
Curr. Opin. Cell Biol.
15
:
23
–30.
Weed, S.A., Y. Du, and J.T. Parsons.
1998
. Translocation of cortactin to the cell periphery is mediated by the small GTPase Rac1.
J. Cell Sci.
111
:
2433
–2443.
Weed, S.A., A.V. Karginov, D.A. Schafer, A.M. Weaver, A.W. Kinley, J.A. Cooper, and J.T. Parsons.
2000
. Cortactin localization to sites of actin assembly in lamellipodia requires interactions with F-actin and the Arp2/3 complex.
J. Cell Biol.
151
:
29
–40.
Woodring, P.J., T. Hunter, and J.Y. Wang.
2003
. Regulation of F-actin-dependent processes by the Abl family of tyrosine kinases.
J. Cell Sci.
116
:
2613
–2626.
Wu, H., and J.T. Parsons.
1993
. Cortactin, an 80/85-kilodalton pp60src substrate, is a filamentous actin-binding protein enriched in the cell cortex.
J. Cell Biol.
120
:
1417
–1426.
Wu, H., A.B. Reynolds, S.B. Kanner, R.R. Vines, and J.T. Parsons.
1991
. Identification and characterization of a novel cytoskeleton-associated pp60src substrate.
Mol. Cell. Biol.
11
:
5113
–5124.
Yano, H., H. Uchida, T. Iwasaki, M. Mukai, H. Akedo, K. Nakamura, S. Hashimoto, and H. Sabe.
2000
. Paxillin α and Crk-associated substrate exert opposing effects on cell migration and contact inhibition of growth through tyrosine phosphorylation.
Proc. Natl. Acad. Sci. USA.
97
:
9076
–9081.

Abbreviations used in this paper: FL, full-length; p130Cas, p130 Crk-associated substrate; SH, Src homology; TM, tyrosine-mutated.

Supplementary data