Activation and Proteasomal Degradation of Rho GTPases by Cytotoxic Necrotizing Factor-1 Elicit a Controlled Inflammatory Response

Abstract

The CNF1 toxin is produced by uropathogenic and meningitis-causing Escherichia coli. CNF1 penetrates autonomously into cells and confers phagocytic properties to epithelial and endothelial cells. CNF1 acts at the molecular level by constitutively activating Rho GTPases attenuated by their cellular ubiquitin-mediated proteasomal degradation. Here we report the relationship between the ubiquitin-mediated proteasomal degradation of activated Rho and the endothelial cell response to the toxin. The type of cellular response to CNF1 intoxication, first screened by DNA microarray analysis, revealed the launching of a program oriented toward an inflammatory response. Parallel to Rho protein activation by CNF1, we also established the kinetics of production of monocyte chemotactic protein-1 (MCP-1), interleukin-8 (IL-8), IL-6, monocyte inflammatory protein-3α (MIP-3α) and E-selectin. Both the mutation of the catalytic domain of the toxin (CNF1-C866S) and the inhibition of Rho proteins abrogate the CNF1-induced production of the immunomodulators MIP-3α, MCP-1, and IL-8. These immunomodulators are also produced upon activation of Cdc42 and Rac preferentially. Our results indicate that, in addition to pathogen molecular pattern recognition by host-receptors, a direct activation of Rho proteins by the CNF1 virulence factor efficiently triggers a cellular reaction of host alert. Consistently, we assume that the CNF1-induced ubiquitin-mediated proteasomal degradation of activated Rho proteins may limit the amplitude of the host cell immune responses. The CNF1 toxin is produced by uropathogenic and meningitis-causing Escherichia coli. CNF1 penetrates autonomously into cells and confers phagocytic properties to epithelial and endothelial cells. CNF1 acts at the molecular level by constitutively activating Rho GTPases attenuated by their cellular ubiquitin-mediated proteasomal degradation. Here we report the relationship between the ubiquitin-mediated proteasomal degradation of activated Rho and the endothelial cell response to the toxin. The type of cellular response to CNF1 intoxication, first screened by DNA microarray analysis, revealed the launching of a program oriented toward an inflammatory response. Parallel to Rho protein activation by CNF1, we also established the kinetics of production of monocyte chemotactic protein-1 (MCP-1), interleukin-8 (IL-8), IL-6, monocyte inflammatory protein-3α (MIP-3α) and E-selectin. Both the mutation of the catalytic domain of the toxin (CNF1-C866S) and the inhibition of Rho proteins abrogate the CNF1-induced production of the immunomodulators MIP-3α, MCP-1, and IL-8. These immunomodulators are also produced upon activation of Cdc42 and Rac preferentially. Our results indicate that, in addition to pathogen molecular pattern recognition by host-receptors, a direct activation of Rho proteins by the CNF1 virulence factor efficiently triggers a cellular reaction of host alert. Consistently, we assume that the CNF1-induced ubiquitin-mediated proteasomal degradation of activated Rho proteins may limit the amplitude of the host cell immune responses. Progress has been made in unraveling the molecular mechanisms of action of bacterial virulence factors, as well as the signal transduction cascades leading to host innate and adaptive immune responses. Studies on host-pathogen interactions are beginning to provide an integrated view of bacterial requirements for successful infection. This is particularly true for a large family of bacterial virulence factors and toxins targeting cellular Rho proteins (1Boquet P. Lemichez E. Trends Cell Biol. 2003; 13: 238-246Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). These bacterial factors either activate or inactivate cellular Rho proteins. Bacterial virulence factors inducing Rho protein activation, among them SopE from Salmonella, IpaC from Shigella, and the cytotoxic necrotizing factor-1 (CNF1) 1The abbreviations used are: CNF1, cytotoxic necrotizing factor-1; HUVECs, human umbilical vein endothelial cells; d-SFM, human endothelial SFM medium (Invitrogen) supplemented with defined growth factors; ELISA, enzyme-linked immunosorbent assay; MAP, mitogen-activated protein; BSA, bovine serum albumin; MCP-1, monocyte chemotactic protein-1; MIP-3α, monocyte inflammatory protein-3α; IL, interleukin; TNF, tumor necrosis factor; GST, glutathione S-transferase; ERK, extracellular signal-regulated kinase; ICAM-1, intercellular adhesion molecule 1; TRAF-1, TNF-receptor associated protein-1; cIAP2, mammalian inhibitor of apoptosis protein-1 homologue C; IM, immunomodulator; RANTES, regulated on activation normal T cell expressed and secreted. from pathogenic Escherichia coli, have all been documented to allow pathogenic bacteria internalization into host cells, despite their different molecular activities (1Boquet P. Lemichez E. Trends Cell Biol. 2003; 13: 238-246Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 2Galan J.E. Zhou D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8754-8761Crossref PubMed Scopus (215) Google Scholar, 3Tran Van Nhieu G. Bourdet-Sicard R. Dumenil G. Blocker A. Sansonetti P.J. Cell Microbiol. 2000; 2: 187-193Crossref PubMed Scopus (115) Google Scholar). Rho proteins are essential regulatory molecules, controlling the actin cytoskeleton organization and dynamic. They act to fine-tune the cell, reshaping it during cell polarity, movement, differentiation, and phagocytosis (4Takai Y. Sasaki T. Matozaki T. Physiol. Rev. 2001; 81: 153-208Crossref PubMed Scopus (2061) Google Scholar, 5Etienne-Manneville S. Hall A. Nature. 2002; 420: 629-635Crossref PubMed Scopus (3854) Google Scholar, 6Chimini G. Chavrier P. Nat. Cell Biol. 2000; 2: 191-196Crossref PubMed Scopus (265) Google Scholar). In addition, they interfere with a variety of signaling pathways controlling gene transcription (7Bishop A.L. Hall A. Biochem. J. 2000; 348: 241-255Crossref PubMed Scopus (1676) Google Scholar). For instance, Rac and Cdc42 are activated down-stream of the Toll-like receptor-2, implicated in Gram-positive pathogen-associated molecular pattern recognition (8Arbibe L. Mira J.P. Teusch N. Kline L. Guha M. Mackman N. Godowski P.J. Ulevitch R.J. Knaus U.G. Nat. Immunol. 2000; 1: 533-540Crossref PubMed Scopus (568) Google Scholar, 9Medzhitov R. Janeway C.A. Science. 2002; 296: 298-300Crossref PubMed Scopus (1672) Google Scholar). Another example of the relationship between Rho proteins and the host defenses is the Rac/Cdc42-dependent formation of a supramolecular activation complex or “immunological synapse” crucial for lymphocyte activation (10Krawczyk C. Penninger J.M. Trends Cell Biol. 2001; 11: 212-220Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). In addition, both Rac1 and Rac2 are required for B-cell development and maintenance (11Walmsley M.J. Ooi S.K. Reynolds L.F. Smith S.H. Ruf S. Mathiot A. Vanes L. Williams D.A. Cancro M.P. Tybulewicz V.L. Science. 2003; 302: 459-462Crossref PubMed Scopus (213) Google Scholar). An important contradiction to be clarified in host-pathogen interactions is the fact that Rho proteins are activated both by bacterial virulence factors and by host cell receptors engaged in pathogen recognition. In this respect, CNF1 toxin is a particularly suitable model. This bacterial toxin specifically deamidates glutamine 63 of RhoA (and its equivalent in Rac and Cdc42) into a glutamic acid (12Flatau G. Lemichez E. Gauthier M. Chardin P. Paris S. Fiorentini C. Boquet P. Nature. 1997; 387: 729-733Crossref PubMed Scopus (425) Google Scholar, 13Schmidt G. Sehr P. Wilm M. Selzer J. Mann M. Aktories K. Nature. 1997; 387: 725-729Crossref PubMed Scopus (464) Google Scholar, 14Lerm M. Schmidt G. Goehring U.M. Schirmer J. Aktories K. J. Biol. Chem. 1999; 274: 28999-29004Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). This point mutation, catalyzed by CNF1, abrogates the GTPase activity of Rho proteins and produces their permanent activation. Once they are constitutively activated, Rho proteins are rapidly conveyed to the ubiquitin-mediated proteasomal degradation pathway (15Doye A. Mettouchi A. Bossis G. Clement R. Buisson-Touati C. Flatau G. Gagnoux L. Piechaczyk M. Boquet P. Lemichez E. Cell. 2002; 111: 553-564Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 16Lerm M. Pop M. Fritz G. Aktories K. Schmidt G. Infect. Immun. 2002; 70: 4053-4058Crossref PubMed Scopus (80) Google Scholar). Thus CNF1 uses a seemingly counterintuitive mechanism, first constitutively activating Rho proteins (deamidation), so they are prone to inactivation (degradation). This mechanism results in a moderate activation of Rho proteins and confers highly invasive properties to CNF1-producing uropathogenic E. coli and meningitis K1 E. coli (15Doye A. Mettouchi A. Bossis G. Clement R. Buisson-Touati C. Flatau G. Gagnoux L. Piechaczyk M. Boquet P. Lemichez E. Cell. 2002; 111: 553-564Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 17Khan N.A. Wang Y. Kim K.J. Chung J.W. Wass C.A. Kim K.S. J. Biol. Chem. 2002; 277: 15607-15612Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). Such tight control of the level of activated Rho proteins is found in other invasive systems. These have been characterized in Salmonella, which co-inject SopE for Rac and Cdc42 activation and SptP for Rac and Cdc42 inactivation (18Kubori T. Galan J.E. Cell. 2003; 115: 333-342Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). As compared with invasive factors injected upon bacterial contact with host cell target, CNF1 shares with the A-B toxins the ability to enter host cells independently of other bacterial factors (1Boquet P. Lemichez E. Trends Cell Biol. 2003; 13: 238-246Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). In the present study we took advantage of the toxic properties of CNF1 to investigate the cellular responses it elicits, independently of the productive bacteria. Cells and Reagents—Human umbilical vein endothelial cells (HUVECs) were obtained from PromoCell (Heidelberg, Germany). Cells were grown in human endothelial SFM medium (Invitrogen) supplemented with defined growth factors (d-SFM): 10 ng/ml epidermal growth factor and 20 ng/ml basic fibroblast growth factor (Invitrogen), 1 μg/ml heparin (Sigma-Aldrich), and either 20% fetal bovine serum (Invitrogen) or 1% (w/v) bovine serum albumin (ELISA grade, Sigma-Aldrich) together with penicillin and streptomycin (Invitrogen). Cells were grown on 0.2% gelatin-coated dishes (Sigma-Aldrich). Transfections of HUVECs were carried out as previously described (19Mettouchi A. Klein S. Guo W. Lopez-Lago M. Lemichez E. Westwick J.K. Giancotti F.G. Mol. Cell. 2001; 8: 115-127Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). Antibodies used were monoclonal anti-β-actin antibody (clone AC-74, Sigma-Aldrich); anti-RhoA, anti-Cdc42, anti-Rac1, and anti-Ras antibodies (BD Transduction Laboratories); anti-HA (clone 11, BabCO); anti-E-selectin (clone CTB202, Santa Cruz Biotechnology); rabbit polyclonal anti-phospho-p44/42 MAP kinase (Thr202/Tyr204), anti-phospsho-p38 MAP kinase (Thr180/Tyr182), and anti-phospho-c-Jun (Ser73, Cell Signaling Technology); anti-human IκBα (Upstate Biotechnology); and anti-TRAF1 (H-186, Santa Cruz Biotechnology). Primary antibodies were visualized using goat anti-mouse or anti-rabbit horseradish peroxidase-conjugated secondary antibodies (DAKO, Glostrup, Denmark). rabbit antibodies were visualized using goat anti-rabbit by horseradish DNA to and were by R. D. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). DNA were by using the R.J. Nat. 1999; PubMed Scopus Google and and as by the HUVECs were at gelatin-coated in HUVECs were in for and in supplemented with HUVECs were in for by Both and of were using the and from used as for the The first and using the (Invitrogen) to the using an a using the and were used in the reaction together with the in transcription the were using at for in a to the in a 10 to for and to for the and The for at at The and in the The to 10 in at by in at The with a at a a in for at by 10 in at An antibody using normal goat as at a of and antibody at a of This by a with a at a of in for at and 10 in at The were at using a The from the were by the E. R. 2002; PubMed Scopus Google Scholar). from the were to in the on as described by the on their the a in level between a and an an or in gene the other the the and of of a were compared expressed as the In analysis, the between the used as the cells, and the the cells by CNF1 for or This of gene by the in a in that a gene or the or CNF1 and toxin E. coli or were grown at in were by in of and using a The for at and the with the of for The and on a The for with the CNF1 with a to during The CNF1 were and on a The CNF1 were and on a a of the with CNF1 using a to CNF1 by The activity of the different of CNF1 toxin by using an cell previously described E. Flatau G. M. Boquet P. Gauthier M. Mol. Microbiol. 1997; PubMed Scopus Google Scholar). The CNF1 toxin used in this study produced at of of cells of the cell activity of the catalytic toxin by its on the CNF1-induced of cells were toxin addition at or well in of cells by addition of medium CNF1, for different of The medium 1 by BSA, for of monocyte chemotactic protein-1 (MCP-1), IL-6, monocyte inflammatory protein-3α and on activation normal T cell expressed and using human as by the and and Cdc42 were as described in A. Mettouchi A. Bossis G. Clement R. Buisson-Touati C. Flatau G. Gagnoux L. Piechaczyk M. Boquet P. Lemichez E. Cell. 2002; 111: 553-564Abstract Full Text Full Text PDF PubMed Scopus (229) Google For activated we made of of as described by the J. 1997; PubMed Scopus Google Scholar). were on and and carried out of Rho GTPases by CNF1 with Cell Signaling molecular mechanism of action of CNF1 is of a permanent activation of Rho which results in their to ubiquitin-mediated proteasomal degradation (12Flatau G. Lemichez E. Gauthier M. Chardin P. Paris S. Fiorentini C. Boquet P. Nature. 1997; 387: 729-733Crossref PubMed Scopus (425) Google Scholar, 13Schmidt G. Sehr P. Wilm M. Selzer J. Mann M. Aktories K. Nature. 1997; 387: 725-729Crossref PubMed Scopus (464) Google Scholar, A. Mettouchi A. Bossis G. Clement R. Buisson-Touati C. Flatau G. Gagnoux L. Piechaczyk M. Boquet P. Lemichez E. Cell. 2002; 111: 553-564Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar, 16Lerm M. Pop M. Fritz G. Aktories K. Schmidt G. Infect. Immun. 2002; 70: 4053-4058Crossref PubMed Scopus (80) Google Scholar). have previously established that this mechanism of action confers invasive properties to pathogenic bacteria (15Doye A. Mettouchi A. Bossis G. Clement R. Buisson-Touati C. Flatau G. Gagnoux L. Piechaczyk M. Boquet P. Lemichez E. Cell. 2002; 111: 553-564Abstract Full Text Full Text PDF PubMed Scopus (229) Google Scholar). that activated Rho proteins interfere with cellular of we the relationship between the host response and the CNF1-induced Rho protein activation and first screened the host cell program by CNF1 in human endothelial cells (HUVECs) using of activation of Rho proteins. that, in addition, this of the type of cellular response engaged by Rho protein activation. HUVECs a cellular for meningitis-causing E. coli CNF1 N.A. Wang Y. Kim K.J. Chung J.W. Wass C.A. Kim K.S. J. Biol. Chem. 2002; 277: 15607-15612Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). In addition, HUVECs have the advantage of cells, an important in signal transduction of CNF1-induced and RhoA activation were first established of the Rho protein family as compared with the These an of the Rho proteins activated by CNF1, other Rho are to the for CNF1 M. Schmidt G. Goehring U.M. Schirmer J. Aktories K. J. Biol. Chem. 1999; 274: 28999-29004Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). indicate that all the Rho proteins a activation to CNF1 The of CNF1 with signaling pathways leading to gene with the of activation, CNF1 and CNF1 to interfere with the protein kinase signaling by and CNF1 also with the as by established that CNF1 pathways to the type of endothelial cell response by CNF1 of Primary Cells by of CNF1 on gene screened both at and the addition of the using and These in a large of the activity of human CNF1 triggers a gene response of a activation of and of the at and and and of the were found to be at and of CNF1 at and of found at a level compared with of CNF1 the kinetics of Rho protein activation by the toxin and be to either engaged specifically at or to In we that the 10 activated a family of inflammatory at and activation and M. P. Immunol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). of inflammatory be to the of the receptors and Cell response to CNF1 also a of cells A. J.M. E. S. A. S. C. J. PubMed Scopus Google Scholar). associated and inhibitor of apoptosis protein-1 homologue were in cell signaling and Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). This in the of the production of a of inflammatory toward the CNF1 activity and Rho protein activation. that, in addition to on the CNF1 mechanism of this this in endothelial of gene activation expressed as the of the gene signal from and cells to the gene signal from control cells signal cell cell that were either or chemotactic and activating chemotactic that have been found different factor cells cell and monocyte family growth T cells innate factor protein factor to innate that have been found different and cytoskeleton for associated that have been found different that have been found different and regulatory and protein regulatory signaling factor of protein response activation that have been found different growth and protein and of factors and regulatory factor 1 factor with growth that have been found different factor of transcription factor of gene transcription factor transcription factor that have been found different acid protein protein protein development and that have been found different cell protein protein protein protein gene that were either or that have been found different in a of gene expressed as the of the gene signal from CNF1 and cells to the gene signal from control cells cell and signaling that were either or necrosis factor cell protein factor factors and transcription of domain protein protein protein that were either or in a of first established the kinetics of production of and IL-6, the and the and of production established by the of CNF1 to of the which found to be of with signaling pathways of between and addition of a level that for The kinetics of and production In addition, we the of production of and in response to CNF1 and with the DNA microarray analysis, we a CNF1-induced production of and at the protein level In we have that HUVECs specifically to the CNF1 toxin by MCP-1, MIP-3α, as well as Rac1 and Cdc42 RhoA the of the of the Rho protein activation by CNF1 for MCP-1, MIP-3α, and first made of toxin from for its properties toward Rho proteins M.J. Aktories K. Rev. Cell Biol. 2002; PubMed Scopus Google Scholar). HUVECs were with different of toxin to CNF1 we that toxin the CNF1-induced production of MCP-1, MIP-3α, and IL-6, in a These results that the activation of Rho proteins by CNF1 is required at for the production of all by This to investigate the of and Cdc42 for of activated Rho protein carried out in of activated Rho proteins by anti-HA production of MCP-1, MIP-3α, and of be in with the level of its by CNF1 of activated Cdc42 or activated Rac1 to the of all the RhoA of Cdc42 and Rac1 for MCP-1, MIP-3α, and in These results are with of the to which CNF1 Rho GTPases in HUVECs, with a activation of Cdc42 and Rac between of Rac and of Rac Cdc42 by CNF1 in the production of a large variety of inflammatory for These results the contradiction of CNF1 as a bacterial virulence factor for the of immune the relationship between the CNF1 molecular mechanism of of Rac activation by proteasomal degradation to the of first established the CNF1-induced production of using different of toxin. production at of by CNF1, to the toxin previously This an of production for cells with of toxin of production were compared with the of CNF1-induced activated of produced were found to with the of activation of Rac by CNF1 These results were also with that of activated Rac in an of production the direct relationship between of activated Rac and the of we the of inhibition toward the CNF1-induced activation of using CNF1 as as we an in the of activated Rac in cells with the inhibitor This from the of of activated results indicate that the cellular inflammatory response from the CNF1-induced activation of Rac may be by the degradation of activated In the present we have the of a relationship between the level of Rho protein activation by CNF1 and its toward a host cell response. As a first we a large of the using to the host cell response at different at the point to a between the of activated Rho proteins and the of of the gene of the were found at both CNF1 were at the protein level and found produced in response to activated Rac or Cdc42 results that the described in a of The cellular response to CNF1 a large of inflammatory for cell and activation. to the of also and family the gene family of which to be by CNF1 Nature. 2003; PubMed Scopus Google and inflammatory as as well as may the of and activation to the of bacterial infection. In with the that cells a response we the of for for pathogen phagocytosis and of the as well as a protein homologue T. C. C. K. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, Y. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). In that MIP-3α, in addition to a for cells, has been to have a activity A. J.M. E. S. A. S. C. J. PubMed Scopus Google Scholar, D. P. J. J. 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This the as to the of and apoptosis during may the of bacteria. of the inflammatory production by CNF1 is that it be by Rho protein to this we that MIP-3α, MCP-1, and were produced upon cellular of activated Cdc42 or activated Rac1 activated In we have that inhibition of using on the CNF1-induced of this of inflammatory results point to Cdc42 and Rac as of the host by CNF1, a of in the of This is by the of the program engaged upon HUVECs to a which produces the activation of Rac (19Mettouchi A. Klein S. Guo W. Lopez-Lago M. Lemichez E. Westwick J.K. Giancotti F.G. Mol. Cell. 2001; 8: 115-127Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, S. P. L. A. Giancotti F.G. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar). In cells a pattern to that of CNF1, ICAM-1, cell adhesion and S. P. L. A. Giancotti F.G. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar). Both that activation, either by for or by CNF1, engaged in is that the of both a cellular response to for pathogen In Cdc42 and in addition to of the cellular response are of the cellular of of in host-pathogen is by both that bacterial virulence factors activate and that Rho proteins are of the host defenses (1Boquet P. Lemichez E. Trends Cell Biol. 2003; 13: 238-246Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Our is to that to the of the engaged in with other invasive bacteria T. C. Sansonetti P.J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). in that of inflammatory are in response to invasive by bacteria. For instance, with this host cells to CNF1 and invasive by specifically and T. C. Sansonetti P.J. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In addition, results a direct relationship between the of Rho protein activation and the amplitude of the host response by cells. it is that bacteria have virulence at a moderate activation of Rho proteins to the cellular program of the host cells. Such a moderate activation of Rho from the activation and inactivation of Rho proteins by invasive factors, may be during of a bacterial bacterial growth and a inflammatory These are in with the development of an by uropathogenic E. coli, which results in an inflammatory In that respect, the mechanism of action of CNF1 of Rho protein activation and proteasomal in a moderate activation of Rho proteins. and for C. toxin and we are to P. P. M. A. and M. M. for of the with