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Rho-activating Escherichia coli cytotoxic necrotizing factor 1: macropinocytosis of apoptotic bodies in human epithelial cells
IJMM IJ
Int. J. Med. Microbiol. 291, 551-554 (2002) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/ijmm
Rho-activating Escherichia coli cytotoxic necrotizing factor 1: macropinocytosis of apoptotic bodies in human epithelial cells Alessia Fabbri1, Loredana Falzano1, Sara Travaglione1, Annarita Stringaro1, Walter Malorni1, Stefano Fais2, Carla Fiorentini1 1 2
Department of Ultrastructures, Istituto Superiore di Sanità, I-00161 Rome, Italy Department of Immunology, Istituto Superiore di Sanità, I-00161 Rome, Italy
Abstract Some pathogenic Escherichia coli strains produce a protein toxin, named cytotoxic necrotizing factor 1 (CNF1), which permanently activates proteins belonging to the Rho family. In epithelial cells, the consequence of this activation is the rearrangement of the actin cytoskeleton and the promotion of an intense and generalized ruffling activity. This leads, in turn, to the induction of a phagocytic-like behavior called macropinocytosis that, in the case of CNF1, depends on the coordinate activation of Rho, Rac and Cdc42. Following internalization, the ingested material is discharged into Rab-7 and Lamp-1-positive acidic vesicles where it probably undergoes degradation. By exerting this activity, CNF1-activated epithelial cells might support the scavenging activity of macrophages during bacterial overgrowth. Key words: macropinocytosis – CNF1 – apoptotic cells – Rho GTPases
Introduction The cytotoxic necrotizing factor type 1 (CNF1) is a 110-kDa monomeric protein toxin produced by some pathogenic strains of Escherichia coli. This toxin permanently activates the small GTPases of the Rho family (Flatau et al., 1997; Schmidt et al., 1997) through deamidation of a pivotal glutamine residue in the switch 2 domain involved in GTP hydrolysis (glutamine 63 in Rho (Flatau et al., 1997; Schmidt et al., 1997) or 61 in Cdc42 and Rac (Lerm et al., 1999)). This leads to the subsequent activation of a number of kinases that control actin-dependent activities such as cell contractility, cell adhesion and spreading (Fiorentini et al., 1997; Lacerda et al., 1997) as well as the promotion of an intense and generalized membrane ruffling that drives macropinocytosis in epithelial cells (Falzano et al., 1993) (reviewed in (Boquet and Fiorentini, 2000)).
Ruffles allow the capture and ingestion of large particles into irregular primary endocytic vesicles, called macropinosomes (Swanson and Watts, 1995), that fuse with lysosomes in professional phagocytes (Racoosin and Swanson, 1993) and recycle back to the cell surface in activated epithelial cells (Hewlett et al., 1994). It has previously been reported that CNF1-treated epithelial cells are capable of exerting a macropinocytotic activity, acquiring the ability to ingest bacteria, either invasive or not, as well as latex particles (Falzano et al., 1993). In the study we herein deal with (Fiorentini et al., 2001), we investigated (i) the specific role of Rho GTPases in the macropinocytosis induced by CNF1 and (ii) the events following the engulfment of apoptotic cells by CNF1-stimulated human epithelial cells. CNF1-triggered macropinocytosis resulted to be driven by the activation of Rho, Rac and Cdc42 and to be followed by a proper degradative pathway.
Corresponding author: Carla Fiorentini, Department of Ultrastructures, Istituto Superiore di Sanità, I-00161 Rome, Italy, Phone: +39 06 49 90 30 06, Fax: +39 06 49 38 71 40, E-mail: [email protected] 1438-4221/01/291/6-7-551 $ 15.00/0
552
A. Fabbri et al.
CNF1-treated human epithelial cells macropinocytose apoptosis-triggered cells but not living cells As prey for phagocyting cells, we used a monomyelocytic cell line, U937 cells, triggered to apoptosis as previously reported (Cossarizza et al., 1995). The staining with the nuclear dye Hoechst allowed the detection of apoptotic bodies in about 40 % of CNF1-treated cells (Fig. 1b) and only 2–3 % of untreated cells (Fig. 1a). The capability of effecting a ruffling-driven capture of the prey, was strongly dependent on the time of exposure to CNF1 (being maximal after 48 h of toxin exposure) and on new protein synthesis (Fiorentini et al., 2001). Intriguingly, CNF1-treated epithelial cells recognized (Fig. 1c) and ingested U937 cells committed to die, irrespectively of the apoptotic stage (measured by annexin V, an early surface marker for cells undergoing apoptosis) whereas they did not react to nor ingest living U937 cells (Fig. 1d).
Rho GTPases are necessary for CNF1induced macropinocytosis in epithelial cells In order to trigger macropinocytosis, CNF1 needs its full enzymatic activity, since a non-toxic mutant of CNF1, in which the catalytic cysteine residue (cys 866) was converted to serine as previously reported (Schmidt et al., 1998), failed in promoting the rufflingdriven capture of apoptotic cells (Fiorentini et al., 2001). The activation of epithelial cells with dominant positive forms of RhoA, Rac and Cdc42 induced a slight but significant (p 0.01) increase in the phagocytic activity when compared to cells transfected with the control plasmid (Fig. 2a). It is evident that DNA transfection per se induced a very modest uptake of apoptotic cells, which is nearly doubled in cells transfected with the GTPases (Fig. 2a). To further investigate the involvement of Rho GTPases in CNF1triggered macropinocytosis, we blocked CNF1 activity by transfecting cells with the dominant negative forms
Fig. 1. Interaction of epithelial HEp-2 cells with U937 cells. Hoechst staining of control (a) and CNF1-treated cells (b) exposed to apoptotic bodies for 3 hours. In toxin-treated cells, apoptotic bodies appeared as a condensed material positive for the nuclear dye. Scanning electron microscopy of CNF1-exposed Hep-2 cells incubated with U937 cells triggered to apoptosis (c) or living cells (d). CNF1-treated cells are able to interact with cells already committed to death only. Scale bars indicate 1 µm.
CNF1-induced macropinocytosis in epithelial cells
553
of Rho GTPases or by using “classical” bacterial protein toxins as inhibitors of Rho proteins. CNF1-treated epithelial cells transfected with the dominant negative forms of Rac (RacN17) or Cdc42 (Cdc42N17) showed a reduced ability to ingest apoptotic bodies (49 3 % and 57 8 %, respectively) with respect to CNF1treated cells transfected with the control plasmid (pcDNA) (Fig. 2b). All the bacterial toxins used (Clostridium botulinum exoenzyme C3, a selective inhibitor of Rho; C. difficile toxin B (CdB), which inactivates Rho, Rac and Cdc42; C. sordellii lethal toxin (LT) which inhibits Ras, Rap, Rac but not Rho or CdC42) significantly impaired the engulfment of apoptotic bodies (Fig. 2c). Altogether, these findings suggest that a coordinate activity of the Rho GTPases is required for the macropinocytotic activity triggered by CNF1.
Macropinosomes in CNF1-activated human epithelial cells may discharge their content into lysosomal degradative compartments We next followed the route undertaken by the captured apoptotic bodies in CNF1-activated epithelial cells. By transmission electron microscopy (TEM) analysis we observed the presence of several apoptotic corps inside cytoplasmic vesicles in CNF1-activated cells (Fig. 3a). A subset of such macropinosomal vesicles containing apoptotic bodies were expressing the late endosomal marker Rab7 (Fiorentini et al., 2001). Moreover, some vesicles displayed a Lamp-1 staining, Lamp-1 being a transmembrane protein specifically associated with lysosomes (Fig. 3b). This finding strongly indicates that macropinosomes in human epithelial cells stimulated by CNF1 may undergo fusion with acidic lysosomallike structures, suggesting the occurrence of a degradative process.
Conclusions We herein show that CNF1-exposed epithelial cells are able to ingest apoptotic but not living cells via a ruffling-driven phenomenon called macropinocytosis. The material, that was captured by a mechanism clearly dependent on the coordinate activation of Rho GTPases, was finally discharged into Rab-7 and Lamp1-positive acidic lysosomal-like vesicles where it probably underwent degradation. CNF1-treated epithelial cells have been reported to aspecifically ingest bacteria, invasive or not, as well as latex particles (Falzano et al., 1993). In the experimental model presented herein, however, the recognition of apoptotic bodies was somehow specific since only
Fig. 2. (a) Percentage of control HEp-2 cells able to macropinocytose apoptotic bodies once transfected with 1 µg of dominant positive forms of Rho GTPase (RhoV14, RacV12, Cdc42V12, myc-tagged). (b) Percentage of CNF1treated epithelial cells able to macropinocytose apoptotic bodies once transfected with dominant negative forms of the Rho GTPases. (c) Inhibition of CNF1-induced macropinocytotic activity by exposure to the Rho-inhibiting bacterial toxins C3B (1 µg/ml), LT (1 µg/ml), CdB (0.5 µg/ml) for 3 h. The results reported as percentages ( standard deviations) of epithelial cells containing apoptotic bodies are from four different experiments in each of which at least 100 cells were counted.
554
A. Fabbri et al.
References
Fig. 3. Intracellular degradation of apoptotic bodies by CNF1-treated epithelial cells. TEM analysis of CNF1-treated cells (a), showing electron-dense apoptotic corps at different stages of degradation inside intracytoplasmatic vesicles (arrowheads). Immunocytochemical analysis of epithelial cells exposed to CNF1 and stained for the lysosomal marker Lamp-1 (b). The arrowhead indicates a Lamp-1-positive vesicle containing an apoptotic cell (stained by TUNEL reaction). Scale bars indicate: (a) 1 µm; (b) 10 µm.
U937 cells committed to die but not living cells were promptly recognized and ingested. This indicates a possible involvement of a receptor in the recognition of apoptotic cells, as also suggested by the need of new protein synthesis for macropinocytosis to occur (Fiorentini et al., 2001). We would like to stress that following the acquisition of the macropinocytotic activity due to CNF1 exposure, epithelial cells may share the job of macrophages, developing the ability to remove apoptotic cells. This scavenging behavior may be normally activated in mucosal epithelial cells in order to support or integrate the activity of resident macrophages during bacterial overgrowth.
Boquet, P., Fiorentini, C.: The cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli. In: Handbook of Experimental Pharmacology: Bacterial Protein Toxins (K. Aktories, I. Just, eds.), Volume 145, pp. 361–379. SpringerVerlag, Heidelberg 2000. Cossarizza, A., Franceschi, C., Monti, D., Salvioli, S., Bellesia, E., Rivabene, R., Biondo, L., Rainaldi, G., Tinari, A., Malorni, W.: Protective effect of N-acetylcysteine in tumor necrosis factor-α-induced apoptosis in U937 cells: the role of mitochondria. Exp. Cell Res. 220, 232–240 (1995). Falzano, L., Fiorentini, C., Donelli, G., Michel, E., Kocks, C., Cossart, P., Cabanié, L., Oswald, E., Boquet, P.: Induction of phagocytic behaviour in human epithelial cells by Escherichia coli cytotoxic necrotizing factor 1. Mol. Microbiol. 9, 1247–1254 (1993). Fiorentini, C., Fabbri, A., Flatau, G., Donelli, G., Matarrese, P., Lemichez, E., Falzano, L., Boquet, P.: Escherichia coli cytotoxic necrotizing factor 1 (CNF1): a toxin which activates the Rho GTPase. J. Biol. Chem. 272, 19 532– 19 537 (1997). Fiorentini, C., Falzano, L., Fabbri, A., Stringaro, A., Lagozzi, M., Travaglione, S., Contamin, S., Arancia, G., Malorni, W., Fais, S.: Activation of Rho GTPases by cytotoxic necrotizing factor 1 induces macropinocytosis and scavenging activity in epithelial cells. Mol. Biol. Cell 12, 2061–2073 (2001). Flatau, G., Lemichez, E., Gauthier, M., Chardin, P., Paris, S., Fiorentini, C. Boquet, P.: Rho GTPase activation by bacterial toxin-induced glutamine deamidation. Nature 387, 729–733 (1997). Hewlett, L. J., Prescott, A. R., Watts, C.: The coated pit and macropinocytotic pathways serve distinct endosome populations. J. Cell Biol. 124, 689–703 (1994). Lacerda, H. M., Pullinger, G. D., Lax, A. J., Rozengurt, E.: Cytotoxic necrotizing factor 1 from Escherichia coli and dermonecrotic toxin from Bordetella bronchiseptica induce p21 Rho-dependent tyrosine phosphorylation of focal adhesion kinase and paxillin in Swiss 3T3 cells. J. Biol. Chem. 272, 9587–9596 (1997). Lerm, M., Selzer, J., Hoffmeyer, A., Rapp, U. R., Aktories, K., Schmidt, G.: Deamidation of Cdc42 and Rac by Escherichia coli cytotoxic necrotizing factor 1: activation of c-Jun N-terminal kinase in HeLa cells. Infect. Immun. 67, 496– 503 (1999). Racoosin, E. L. Swanson, J. A. : Macropinosome maturation and fusion with tubular lysosomes in macrophages. J. Cell Biol. 121, 1011–1020 (1993). Schmidt, G., Sehr, P., Wilm, M., Selzer, J., Mann, M., Aktories, K.: Gln 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor 1. Nature 387, 725–729 (1997). Schmidt, G., Selzer, J., Lerm, M., Aktories, K.: The Rhodeamidating cytotoxic necrotizing factor 1 from Escherichia coli possesses transglutaminase activity. J. Biol. Chem. 273, 13 669–13 674 (1998). Swanson, J. A., Watts, C.: Macropinocytosis. Trends Cell Biol. 5, 424–428 (1995).
Int. J. Med. Microbiol. 291, 551-554 (2002) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/ijmm
Rho-activating Escherichia coli cytotoxic necrotizing factor 1: macropinocytosis of apoptotic bodies in human epithelial cells Alessia Fabbri1, Loredana Falzano1, Sara Travaglione1, Annarita Stringaro1, Walter Malorni1, Stefano Fais2, Carla Fiorentini1 1 2
Department of Ultrastructures, Istituto Superiore di Sanità, I-00161 Rome, Italy Department of Immunology, Istituto Superiore di Sanità, I-00161 Rome, Italy
Abstract Some pathogenic Escherichia coli strains produce a protein toxin, named cytotoxic necrotizing factor 1 (CNF1), which permanently activates proteins belonging to the Rho family. In epithelial cells, the consequence of this activation is the rearrangement of the actin cytoskeleton and the promotion of an intense and generalized ruffling activity. This leads, in turn, to the induction of a phagocytic-like behavior called macropinocytosis that, in the case of CNF1, depends on the coordinate activation of Rho, Rac and Cdc42. Following internalization, the ingested material is discharged into Rab-7 and Lamp-1-positive acidic vesicles where it probably undergoes degradation. By exerting this activity, CNF1-activated epithelial cells might support the scavenging activity of macrophages during bacterial overgrowth. Key words: macropinocytosis – CNF1 – apoptotic cells – Rho GTPases
Introduction The cytotoxic necrotizing factor type 1 (CNF1) is a 110-kDa monomeric protein toxin produced by some pathogenic strains of Escherichia coli. This toxin permanently activates the small GTPases of the Rho family (Flatau et al., 1997; Schmidt et al., 1997) through deamidation of a pivotal glutamine residue in the switch 2 domain involved in GTP hydrolysis (glutamine 63 in Rho (Flatau et al., 1997; Schmidt et al., 1997) or 61 in Cdc42 and Rac (Lerm et al., 1999)). This leads to the subsequent activation of a number of kinases that control actin-dependent activities such as cell contractility, cell adhesion and spreading (Fiorentini et al., 1997; Lacerda et al., 1997) as well as the promotion of an intense and generalized membrane ruffling that drives macropinocytosis in epithelial cells (Falzano et al., 1993) (reviewed in (Boquet and Fiorentini, 2000)).
Ruffles allow the capture and ingestion of large particles into irregular primary endocytic vesicles, called macropinosomes (Swanson and Watts, 1995), that fuse with lysosomes in professional phagocytes (Racoosin and Swanson, 1993) and recycle back to the cell surface in activated epithelial cells (Hewlett et al., 1994). It has previously been reported that CNF1-treated epithelial cells are capable of exerting a macropinocytotic activity, acquiring the ability to ingest bacteria, either invasive or not, as well as latex particles (Falzano et al., 1993). In the study we herein deal with (Fiorentini et al., 2001), we investigated (i) the specific role of Rho GTPases in the macropinocytosis induced by CNF1 and (ii) the events following the engulfment of apoptotic cells by CNF1-stimulated human epithelial cells. CNF1-triggered macropinocytosis resulted to be driven by the activation of Rho, Rac and Cdc42 and to be followed by a proper degradative pathway.
Corresponding author: Carla Fiorentini, Department of Ultrastructures, Istituto Superiore di Sanità, I-00161 Rome, Italy, Phone: +39 06 49 90 30 06, Fax: +39 06 49 38 71 40, E-mail: [email protected] 1438-4221/01/291/6-7-551 $ 15.00/0
552
A. Fabbri et al.
CNF1-treated human epithelial cells macropinocytose apoptosis-triggered cells but not living cells As prey for phagocyting cells, we used a monomyelocytic cell line, U937 cells, triggered to apoptosis as previously reported (Cossarizza et al., 1995). The staining with the nuclear dye Hoechst allowed the detection of apoptotic bodies in about 40 % of CNF1-treated cells (Fig. 1b) and only 2–3 % of untreated cells (Fig. 1a). The capability of effecting a ruffling-driven capture of the prey, was strongly dependent on the time of exposure to CNF1 (being maximal after 48 h of toxin exposure) and on new protein synthesis (Fiorentini et al., 2001). Intriguingly, CNF1-treated epithelial cells recognized (Fig. 1c) and ingested U937 cells committed to die, irrespectively of the apoptotic stage (measured by annexin V, an early surface marker for cells undergoing apoptosis) whereas they did not react to nor ingest living U937 cells (Fig. 1d).
Rho GTPases are necessary for CNF1induced macropinocytosis in epithelial cells In order to trigger macropinocytosis, CNF1 needs its full enzymatic activity, since a non-toxic mutant of CNF1, in which the catalytic cysteine residue (cys 866) was converted to serine as previously reported (Schmidt et al., 1998), failed in promoting the rufflingdriven capture of apoptotic cells (Fiorentini et al., 2001). The activation of epithelial cells with dominant positive forms of RhoA, Rac and Cdc42 induced a slight but significant (p 0.01) increase in the phagocytic activity when compared to cells transfected with the control plasmid (Fig. 2a). It is evident that DNA transfection per se induced a very modest uptake of apoptotic cells, which is nearly doubled in cells transfected with the GTPases (Fig. 2a). To further investigate the involvement of Rho GTPases in CNF1triggered macropinocytosis, we blocked CNF1 activity by transfecting cells with the dominant negative forms
Fig. 1. Interaction of epithelial HEp-2 cells with U937 cells. Hoechst staining of control (a) and CNF1-treated cells (b) exposed to apoptotic bodies for 3 hours. In toxin-treated cells, apoptotic bodies appeared as a condensed material positive for the nuclear dye. Scanning electron microscopy of CNF1-exposed Hep-2 cells incubated with U937 cells triggered to apoptosis (c) or living cells (d). CNF1-treated cells are able to interact with cells already committed to death only. Scale bars indicate 1 µm.
CNF1-induced macropinocytosis in epithelial cells
553
of Rho GTPases or by using “classical” bacterial protein toxins as inhibitors of Rho proteins. CNF1-treated epithelial cells transfected with the dominant negative forms of Rac (RacN17) or Cdc42 (Cdc42N17) showed a reduced ability to ingest apoptotic bodies (49 3 % and 57 8 %, respectively) with respect to CNF1treated cells transfected with the control plasmid (pcDNA) (Fig. 2b). All the bacterial toxins used (Clostridium botulinum exoenzyme C3, a selective inhibitor of Rho; C. difficile toxin B (CdB), which inactivates Rho, Rac and Cdc42; C. sordellii lethal toxin (LT) which inhibits Ras, Rap, Rac but not Rho or CdC42) significantly impaired the engulfment of apoptotic bodies (Fig. 2c). Altogether, these findings suggest that a coordinate activity of the Rho GTPases is required for the macropinocytotic activity triggered by CNF1.
Macropinosomes in CNF1-activated human epithelial cells may discharge their content into lysosomal degradative compartments We next followed the route undertaken by the captured apoptotic bodies in CNF1-activated epithelial cells. By transmission electron microscopy (TEM) analysis we observed the presence of several apoptotic corps inside cytoplasmic vesicles in CNF1-activated cells (Fig. 3a). A subset of such macropinosomal vesicles containing apoptotic bodies were expressing the late endosomal marker Rab7 (Fiorentini et al., 2001). Moreover, some vesicles displayed a Lamp-1 staining, Lamp-1 being a transmembrane protein specifically associated with lysosomes (Fig. 3b). This finding strongly indicates that macropinosomes in human epithelial cells stimulated by CNF1 may undergo fusion with acidic lysosomallike structures, suggesting the occurrence of a degradative process.
Conclusions We herein show that CNF1-exposed epithelial cells are able to ingest apoptotic but not living cells via a ruffling-driven phenomenon called macropinocytosis. The material, that was captured by a mechanism clearly dependent on the coordinate activation of Rho GTPases, was finally discharged into Rab-7 and Lamp1-positive acidic lysosomal-like vesicles where it probably underwent degradation. CNF1-treated epithelial cells have been reported to aspecifically ingest bacteria, invasive or not, as well as latex particles (Falzano et al., 1993). In the experimental model presented herein, however, the recognition of apoptotic bodies was somehow specific since only
Fig. 2. (a) Percentage of control HEp-2 cells able to macropinocytose apoptotic bodies once transfected with 1 µg of dominant positive forms of Rho GTPase (RhoV14, RacV12, Cdc42V12, myc-tagged). (b) Percentage of CNF1treated epithelial cells able to macropinocytose apoptotic bodies once transfected with dominant negative forms of the Rho GTPases. (c) Inhibition of CNF1-induced macropinocytotic activity by exposure to the Rho-inhibiting bacterial toxins C3B (1 µg/ml), LT (1 µg/ml), CdB (0.5 µg/ml) for 3 h. The results reported as percentages ( standard deviations) of epithelial cells containing apoptotic bodies are from four different experiments in each of which at least 100 cells were counted.
554
A. Fabbri et al.
References
Fig. 3. Intracellular degradation of apoptotic bodies by CNF1-treated epithelial cells. TEM analysis of CNF1-treated cells (a), showing electron-dense apoptotic corps at different stages of degradation inside intracytoplasmatic vesicles (arrowheads). Immunocytochemical analysis of epithelial cells exposed to CNF1 and stained for the lysosomal marker Lamp-1 (b). The arrowhead indicates a Lamp-1-positive vesicle containing an apoptotic cell (stained by TUNEL reaction). Scale bars indicate: (a) 1 µm; (b) 10 µm.
U937 cells committed to die but not living cells were promptly recognized and ingested. This indicates a possible involvement of a receptor in the recognition of apoptotic cells, as also suggested by the need of new protein synthesis for macropinocytosis to occur (Fiorentini et al., 2001). We would like to stress that following the acquisition of the macropinocytotic activity due to CNF1 exposure, epithelial cells may share the job of macrophages, developing the ability to remove apoptotic cells. This scavenging behavior may be normally activated in mucosal epithelial cells in order to support or integrate the activity of resident macrophages during bacterial overgrowth.
Boquet, P., Fiorentini, C.: The cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli. In: Handbook of Experimental Pharmacology: Bacterial Protein Toxins (K. Aktories, I. Just, eds.), Volume 145, pp. 361–379. SpringerVerlag, Heidelberg 2000. Cossarizza, A., Franceschi, C., Monti, D., Salvioli, S., Bellesia, E., Rivabene, R., Biondo, L., Rainaldi, G., Tinari, A., Malorni, W.: Protective effect of N-acetylcysteine in tumor necrosis factor-α-induced apoptosis in U937 cells: the role of mitochondria. Exp. Cell Res. 220, 232–240 (1995). Falzano, L., Fiorentini, C., Donelli, G., Michel, E., Kocks, C., Cossart, P., Cabanié, L., Oswald, E., Boquet, P.: Induction of phagocytic behaviour in human epithelial cells by Escherichia coli cytotoxic necrotizing factor 1. Mol. Microbiol. 9, 1247–1254 (1993). Fiorentini, C., Fabbri, A., Flatau, G., Donelli, G., Matarrese, P., Lemichez, E., Falzano, L., Boquet, P.: Escherichia coli cytotoxic necrotizing factor 1 (CNF1): a toxin which activates the Rho GTPase. J. Biol. Chem. 272, 19 532– 19 537 (1997). Fiorentini, C., Falzano, L., Fabbri, A., Stringaro, A., Lagozzi, M., Travaglione, S., Contamin, S., Arancia, G., Malorni, W., Fais, S.: Activation of Rho GTPases by cytotoxic necrotizing factor 1 induces macropinocytosis and scavenging activity in epithelial cells. Mol. Biol. Cell 12, 2061–2073 (2001). Flatau, G., Lemichez, E., Gauthier, M., Chardin, P., Paris, S., Fiorentini, C. Boquet, P.: Rho GTPase activation by bacterial toxin-induced glutamine deamidation. Nature 387, 729–733 (1997). Hewlett, L. J., Prescott, A. R., Watts, C.: The coated pit and macropinocytotic pathways serve distinct endosome populations. J. Cell Biol. 124, 689–703 (1994). Lacerda, H. M., Pullinger, G. D., Lax, A. J., Rozengurt, E.: Cytotoxic necrotizing factor 1 from Escherichia coli and dermonecrotic toxin from Bordetella bronchiseptica induce p21 Rho-dependent tyrosine phosphorylation of focal adhesion kinase and paxillin in Swiss 3T3 cells. J. Biol. Chem. 272, 9587–9596 (1997). Lerm, M., Selzer, J., Hoffmeyer, A., Rapp, U. R., Aktories, K., Schmidt, G.: Deamidation of Cdc42 and Rac by Escherichia coli cytotoxic necrotizing factor 1: activation of c-Jun N-terminal kinase in HeLa cells. Infect. Immun. 67, 496– 503 (1999). Racoosin, E. L. Swanson, J. A. : Macropinosome maturation and fusion with tubular lysosomes in macrophages. J. Cell Biol. 121, 1011–1020 (1993). Schmidt, G., Sehr, P., Wilm, M., Selzer, J., Mann, M., Aktories, K.: Gln 63 of Rho is deamidated by Escherichia coli cytotoxic necrotizing factor 1. Nature 387, 725–729 (1997). Schmidt, G., Selzer, J., Lerm, M., Aktories, K.: The Rhodeamidating cytotoxic necrotizing factor 1 from Escherichia coli possesses transglutaminase activity. J. Biol. Chem. 273, 13 669–13 674 (1998). Swanson, J. A., Watts, C.: Macropinocytosis. Trends Cell Biol. 5, 424–428 (1995).