Cytotoxic necrotizing factors (CNFs) encompass a class of autotransporter toxins produced by uropathogenic E. coli (CNF1) or Y. pseudotuberculosis (CNFy). CNF toxins deamidate and thereby constitutively activate RhoA, Rac1, and Cdc42. In this study, the effects of CNF1 on cell-matrix adhesion are analysed using functional cell-adhesion assays. CNF1 strongly increased cell-matrix binding of suspended Hela cells and decreased the susceptibly of cells to trypsin-induced cell detachment. Increased cell-matrix binding was also observed upon treatment of Hela cells with isomeric CNFy, that specifically deamidates RhoA. Increased cell-matrix binding thus appears to depend on RhoA deamidation. In contrast, increased cell spreading was specifically observed upon CNF1 treatment, suggesting that it rather depended on Rac1/Cdc42 deamidation. Increased cell-matrix adhesion is further presented to result in reduced cell migration of adherent cells. In contrast, migration of suspended cells was not affected upon treatment with CNF1 or CNFy. CNF1 and CNFy thus reduced cell migration specifically under the condition of pre-established cell-matrix adhesion. 1. Introduction Cell-matrix adhesion involves several processes including integrin binding, cell spreading, and flattening against the substrate. Cultured cells, that spread out on ligand coated surfaces, rearrange their cytoskeleton and begin to move. Integrins thereby cluster together in “focal complexes” at the leading edge. These focal complexes grow into mature focal contacts, also called focal adhesions (FAs) [1]. Focal adhesions contain over 100 different proteins, including integrins, adapter proteins, and intracellular signaling proteins. Clustered integrins anchor actin filaments to the cell membrane and link them with the extracellular matrix (ECM) through adapter proteins such as talin and vinculin. The adapter protein paxillin links integrins to signaling proteins, forming a scaffold for Src kinases, the focal adhesion kinase (FAK), or the p21-activated kinase (PAK) [2–5]. The turnover of FAs in moving cells is driven by small GTPases of the Rho subfamily. FA formation and disassembly at the leading edge is driven by Rac1 and the localized suppression of Rho activity. Disassembly of FAs at the cell rear requires RhoA activity [6]. The activity of Rho proteins is regulated by the GTPase cycle. Rho proteins are active in the GTP-bound state and inactive in the GDP-bound state. In their active conformation Rho proteins interact with effector proteins to transmit downstream signaling. The cycling between
References
[1]
K. Bhadriraju, M. Yang, S. A. Ruiz, D. Pirone, J. Tan, and C. S. Chen, “Activation of ROCK by RhoA is regulated by cell adhesion, shape, and cytoskeletal tension,” Experimental Cell Research, vol. 313, no. 16, pp. 3616–3623, 2007.
[2]
A. L. Berrier and K. M. Yamada, “Cell-matrix adhesion,” Journal of Cellular Physiology, vol. 213, no. 3, pp. 565–573, 2007.
[3]
P. M. Chan, L. Lim, and E. Manser, “PAK is regulated by PI3K, PIX, CDC42, and PP2Cα and mediates focal adhesion turnover in the hyperosmotic stress-induced p38 pathway,” Journal of Biological Chemistry, vol. 283, no. 36, pp. 24949–24961, 2008.
[4]
D. J. Webb, J. T. Parsons, and A. F. Horwitz, “Adhesion assembly, disassembly and turnover in migrating cells—over and over and over again,” Nature Cell Biology, vol. 4, no. 4, pp. E97–E100, 2002.
[5]
J. P. T. Klooster, Z. M. Jaffer, J. Chernoff, and P. L. Hordijk, “Targeting and activation of Rac1 are mediated by the exchange factor β-Pix,” Journal of Cell Biology, vol. 172, no. 5, pp. 759–769, 2006.
[6]
J. T. Parsons, A. R. Horwitz, and M. A. Schwartz, “Cell adhesion: integrating cytoskeletal dynamics and cellular tension,” Nature Reviews Molecular Cell Biology, vol. 11, no. 9, pp. 633–643, 2010.
[7]
M. Lemonnier, L. Landraud, and E. Lemichez, “Rho GTPase-activating bacterial toxins: from bacterial virulence regulation to eukaryotic cell biology,” FEMS Microbiology Reviews, vol. 31, no. 5, pp. 515–534, 2007.
[8]
Z. Knust and G. Schmidt, “Cytotoxic necrotizing factors (CNFs)-a growing toxin family,” Toxins, vol. 2, no. 1, pp. 116–127, 2010.
[9]
A. Fabbri, S. Travaglione, and C. Fiorentini, “Escherichia coli cytotoxic necrotizing factor 1 (CNF1): toxin biology, in vivo applications and therapeutic potential,” Toxins, vol. 2, no. 2, pp. 283–296, 2010.
[10]
C. Capo, S. Meconi, M. V. Sanguedolce et al., “Effect of cytotoxic necrotizing factor-1 on actin cytoskeleton in human monocytes: role in the regulation of integrin-dependent phagocytosis,” Journal of Immunology, vol. 161, no. 8, pp. 4301–4308, 1998.
[11]
C. Fiorentini, A. Fabbri, G. Flatau et al., “Escherichia coli cytotoxic necrotizing factor 1 (CNF1), a toxin that activates the Rho GTPase,” Journal of Biological Chemistry, vol. 272, no. 31, pp. 19532–19537, 1997.
[12]
H. M. Lacerda, G. D. Pullinger, A. J. Lax, and E. Rozengurt, “Cytotoxic necrotizing factor 1 from Escherichia coli and dermonecrotic toxin from Bordetella bronchiseptica induce -dependent tyrosine phosphorylation of focal adhesion kinase and paxillin in Swiss 3T3 cells,” Journal of Biological Chemistry, vol. 272, no. 14, pp. 9587–9596, 1997.
[13]
C. Florentini, P. Matarrese, E. Straface et al., “Toxin-induced activation of Rho GTP-binding protein increases Bcl-2 expression and influences mitochondrial homeostasis,” Experimental Cell Research, vol. 242, no. 1, pp. 341–350, 1998.
[14]
S. C. Huelsenbeck, M. May, G. Schmidt, and H. Genth, “Inhibition of cytokinesis by Clostridium difficile toxin B and cytotoxic necrotizing factors—reinforcing the critical role of RhoA in cytokinesis,” Cell Motility and the Cytoskeleton, vol. 66, no. 11, pp. 967–975, 2009.
[15]
C. Hoffmann, M. Pop, J. Leemhuis, J. Schirmer, K. Aktories, and G. Schmidt, “The Yersinia pseudotuberculosis cytotoxic necrotizing factor ( ) selectively activates RhoA,” Journal of Biological Chemistry, vol. 279, no. 16, pp. 16026–16032, 2004.
[16]
J. Huelsenbeck, S. C. Dreger, R. Gerhard, G. Fritz, I. Just, and H. Genth, “Upregulation of the immediate early gene product RhoB by exoenzyme C3 from Clostridium limosum and toxin B from Clostridium difficile,” Biochemistry, vol. 46, no. 16, pp. 4923–4931, 2007.
[17]
G. Schmidt, P. Sehr, M. Wilm, J. Seizer, M. Mann, and K. Aktories, “Gin 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor-1,” Nature, vol. 387, no. 6634, pp. 725–729, 1997.
[18]
A. Doye, A. Mettouchi, G. Bossis et al., “CNF1 exploits the ubiquitin-proteasome machinery to restrict Rho GTPase activation for bacterial host cell invasion,” Cell, vol. 111, no. 4, pp. 553–564, 2002.
[19]
M. Lerm, M. Pop, G. Fritz, K. Aktories, and G. Schmidt, “Proteasomal degradation of cytotoxic necrotizing factor 1-activated Rac,” Infection and Immunity, vol. 70, no. 8, pp. 4053–4058, 2002.
[20]
H. Genth, S. C. Dreger, J. Huelsenbeck, and I. Just, “Clostridium difficile toxins: more than mere inhibitors of Rho proteins,” International Journal of Biochemistry and Cell Biology, vol. 40, no. 4, pp. 592–597, 2008.
[21]
J. Huelsenbeck, S. Dreger, R. Gerhard, H. Barth, I. Just, and H. Genth, “Difference in the cytotoxic effects of toxin B from Clostridium difficile strain VPI 10463 and toxin B from variant Clostridium difficile strain 1470,” Infection and Immunity, vol. 75, no. 2, pp. 801–809, 2007.
[22]
P. B. van Hennik, J. P. T. Klooster, J. R. Halstead et al., “The C-terminal domain of Rac1 contains two motifs that control targeting and signaling specificity,” Journal of Biological Chemistry, vol. 278, no. 40, pp. 39166–39175, 2003.
[23]
D. Riveline, E. Zamir, N. Q. Balaban et al., “Focal contacts as mechanosensors: externally applied local mechanical force induces growth of focal contacts by an mDia1-dependent and ROCK-independent mechanism,” Journal of Cell Biology, vol. 153, no. 6, pp. 1175–1185, 2001.
[24]
Z. S. Zhao, E. Manser, T. H. Loo, and L. Lim, “Coupling of PAK-interacting exchange factor PIX to GIT1 promotes focal complex disassembly,” Molecular and Cellular Biology, vol. 20, no. 17, pp. 6354–6363, 2000.
[25]
F. P. L. Lai, M. Szczodrak, J. M. Oelkers et al., “Cortactin promotes migration and platelet-derived growth factor-induced actin reorganization by signaling to Rho-GTPases,” Molecular Biology of the Cell, vol. 20, no. 14, pp. 3209–3223, 2009.
[26]
M. Z. Gilcrease, “Integrin signaling in epithelial cells,” Cancer Letters, vol. 247, no. 1-2, pp. 1–25, 2007.
[27]
S. J. Coniglio, T. S. Jou, and M. Symons, “Rac1 protects epithelial cells against anoikis,” Journal of Biological Chemistry, vol. 276, no. 30, pp. 28113–28120, 2001.
[28]
C. Fiorentini, P. Matarrese, E. Straface et al., “Rho-dependent cell spreading activated by E.coli cytotoxic necrotizing factor 1 hinders apoptosis in epithelial cells,” Cell Death and Differentiation, vol. 5, no. 11, pp. 921–929, 1998.
[29]
A. G. Miraglia, S. Travaglione, S. Meschini et al., “Cytotoxic necrotizing factor 1 prevents apoptosis via the Akt/IκB kinase pathway: role of nuclear factor-κB and Bcl-2,” Molecular Biology of the Cell, vol. 18, no. 7, pp. 2735–2744, 2007.
[30]
T. L. Cheng, M. Symons, and T. S. Jou, “Regulation of anoikis by Cdc42 and Rac1,” Experimental Cell Research, vol. 295, no. 2, pp. 497–511, 2004.
