IpgB1 Is a Novel Shigella Effector Protein Involved in Bacterial Invasion of Host Cells

Abstract

Shigella, the causative agent of bacillary dysentery, is capable of inducing the large scale membrane ruffling required for the bacterial invasion of host cells. Shigella secrete a subset of effectors via the type III secretion system (TTSS) into the host cells to induce membrane ruffling. Here, we show that IpgB1 is secreted via the TTSS into epithelial cells and plays a major role in producing membrane ruffles via stimulation of Rac1 and Cdc42 activities, thus promoting bacterial invasion of epithelial cells. The invasiveness of the ipgB1 mutant was decreased to less than 50% of the wild-type level (100%) in a gentamicin protection or plaque forming assay. HeLa cells infected with the wild-type or a IpgB1-hyperproducing strain developed membrane ruffles, with the invasiveness and the scale of membrane ruffles being comparable with the level of IpgB1 production in bacteria. Upon expression of EGFP-IpgB1 in HeLa cells, large membrane ruffles are extended, where the EGFP-IpgB1 was predominantly associated with the cytoplasmic membrane. The IpgB1-mediated formation of ruffles was significantly diminished by expressing Rac1 small interfering RNA and Cdc42 small interfering RNA or by treatment with GGTI-298, an inhibitor of the geranylgeranylation of Rho GTPases. When IpgB1 was expressed in host cells or wild-type Shigella-infected host cells, Rac1 and Cdc42 were activated. The results thus indicate that IpgB1 is a novel Shigella effector involved in bacterial invasion of epithelial cells via the activation of Rho GTPases. Shigella, the causative agent of bacillary dysentery, is capable of inducing the large scale membrane ruffling required for the bacterial invasion of host cells. Shigella secrete a subset of effectors via the type III secretion system (TTSS) into the host cells to induce membrane ruffling. Here, we show that IpgB1 is secreted via the TTSS into epithelial cells and plays a major role in producing membrane ruffles via stimulation of Rac1 and Cdc42 activities, thus promoting bacterial invasion of epithelial cells. The invasiveness of the ipgB1 mutant was decreased to less than 50% of the wild-type level (100%) in a gentamicin protection or plaque forming assay. HeLa cells infected with the wild-type or a IpgB1-hyperproducing strain developed membrane ruffles, with the invasiveness and the scale of membrane ruffles being comparable with the level of IpgB1 production in bacteria. Upon expression of EGFP-IpgB1 in HeLa cells, large membrane ruffles are extended, where the EGFP-IpgB1 was predominantly associated with the cytoplasmic membrane. The IpgB1-mediated formation of ruffles was significantly diminished by expressing Rac1 small interfering RNA and Cdc42 small interfering RNA or by treatment with GGTI-298, an inhibitor of the geranylgeranylation of Rho GTPases. When IpgB1 was expressed in host cells or wild-type Shigella-infected host cells, Rac1 and Cdc42 were activated. The results thus indicate that IpgB1 is a novel Shigella effector involved in bacterial invasion of epithelial cells via the activation of Rho GTPases. IntroductionShigella are highly adapted human pathogens and are the cause of bacillary dysentery. The prominent pathogenic feature of Shigella is the ability to invade a variety of cells during infection of the intestinal mucosa, including enterocytes, macrophages, dendritic cells, and neutrophils. When Shigella reach the colon, they translocate through the epithelial barrier via the M cells that overlie solitary lymphoid nodules. Once they reach the underlying M cells, Shigella invade the resident macrophages, and the infecting bacteria escape from the phagosome into the cytoplasm. Shigella multiply in the macrophage cytoplasm and finally induce cell death (1Zychlinsky A. Prevost M.C. Sansonetti P.J. Nature. 1992; 358: 167-169Crossref PubMed Scopus (775) Google Scholar, 2Suzuki T. Nakanishi K. Tsutsui H. Iwai H. Akira S. Inohara N. Chamaillard M. Nuñez G. Sasakawa C. J. Biol. Chem. 2005; 280: 14042-14050Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Shigella released from the dead macrophages enter the surrounding enterocytes through their basolateral surface by inducing large scale membrane ruffling. As soon as a bacterium is surrounded by a membrane vacuole, it immediately disrupts the vacuole membrane and escapes into the cytoplasm. Shigella multiply in the cytoplasm and move about by inducing actin polymerization at one pole of the bacterium, by which the pathogen moves within the cytoplasm as well as into the adjacent cells (3Cossart P. Sansonetti P.J. Science. 2004; 304: 242-248Crossref PubMed Scopus (786) Google Scholar). Thus, the ability of bacteria to enter the colonic epithelial cells is essential for colonization within the intestinal epithelium.For bacterial invasion of epithelial cells, Shigella (and Salmonella) use a special mechanism called the “trigger mechanism of entry,” which is characterized by macropinocytic and phagocytic events that allow cells to trap several bacterial particles simultaneously (4Sasakawa C. Lamont R. Bacterial Invasion of Host Cells. 5. Cambridge University Press, Cambridge, UK2004: 25-57Google Scholar). When Shigella comes into contact with epithelial cells, the type III secretion system (TTSS) 1The abbreviations used are: TTSS, type III secretion system; GST, glutathione S-transferase; CRIB, Cdc42/Rac-interactive binding domain; TRITC, tetramethylrhodamine isothiocyanate; FCS, fetal calf serum; CR, Congo red; PBS, phosphate-buffered saline; IPTG, isopropyl-1-thio-β-d-galactopyranoside; MOI, multiplicity of infection; GGTI, geranylgeranyltransferase inhibitor; FTI, farnesyltransferase inhibitor; siRNA, small interfering RNA; MDCK, Madin-Darby canine kidney; WT, wild type; EGFP, enhanced green fluorescent protein; LPS, lipopolysaccharide. is stimulated and delivers the effectors into the host cells and surrounding bacterial space (5Blocker A. Komoriya K. Aizawa S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3027-3030Crossref PubMed Scopus (242) Google Scholar). The secreted effectors are capable of modulating various host functions engaged in remodeling the surface architecture of the host cell and escape from the host innate defense systems (4Sasakawa C. Lamont R. Bacterial Invasion of Host Cells. 5. Cambridge University Press, Cambridge, UK2004: 25-57Google Scholar, 6Ogawa M. Yoshimori T. Suzuki T. Sagara H. Mizushima N. Sasakawa C. Science. 2005; 307: 727-731Crossref PubMed Scopus (694) Google Scholar). Studies have indicated that several Shigella effectors, including IpaA, IpaB, IpaC, IpgD, and VirA, secreted via the TTSS are involved in stimulating the reorganization of F-actin and microtubule cytoskeletons that trigger the bacterial uptake by host cells. IpaB, for example, is capable of binding to the hyaluronic acid receptor CD44 (7Skoudy A. Mounier J. Aruffo A. Ohayon H. Gounon P. Sansonetti P. Tran Van Nhieu G. Cell Microbiol. 2000; 2: 19-33Crossref PubMed Scopus (105) Google Scholar). The IpaB-CD44 binding occurring within rafts (8van der Goot F.G. Tran Van Nhieu G. Allaoui A. Sansonetti P. Lafont F. J. Biol. Chem. 2004; 279: 47792-47798Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 9Lafont F. Tran Van Nhieu G. Hanada K. Sansonetti P. van der Goot F.G. EMBO J. 2002; 21: 4449-4457Crossref PubMed Scopus (193) Google Scholar) is capable of stimulating the cell signaling involved in promoting Shigella invasion via the accumulation of c-Src and stimulation of the phosphorylation of cortactin and Crk with the aid of the Abl tyrosine kinases (10Burton E.A. Plattner R. Pendergast A.M. EMBO J. 2003; 22: 5471-5479Crossref PubMed Scopus (109) Google Scholar, 11Tran Van Nhieu G. Enninga J. Sansonetti P. Grompone G. Curr. Opin. Microbiol. 2005; 8: 16-20Crossref PubMed Scopus (48) Google Scholar, 12Duménil G. Sansonetti P. Tran Van Nhieu G. J. Cell Sci. 2000; 113: 71-80PubMed Google Scholar, 13Bougnères L. Girardin S.E. Weed S.A. Karginov A.V. Olivo-Marin J.C. Parsons J.T. Sansonetti P.J. Tran Van Nhieu G. J. Cell Biol. 2004; 166: 225-235Crossref PubMed Scopus (73) Google Scholar). IpaC is capable of leading to the activation of Cdc42 and Rac1, since IpaC can be integrated into the host cytoplasmic membrane, which somehow leads to the recruitment of cortactin and activation of c-Src, thus promoting local actin polymerization (3Cossart P. Sansonetti P.J. Science. 2004; 304: 242-248Crossref PubMed Scopus (786) Google Scholar, 11Tran Van Nhieu G. Enninga J. Sansonetti P. Grompone G. Curr. Opin. Microbiol. 2005; 8: 16-20Crossref PubMed Scopus (48) Google Scholar, 14Tran Van Nhieu G. Caron E. Hall A. Sansonetti P.J. EMBO J. 1999; 18: 3249-3262Crossref PubMed Scopus (202) Google Scholar). VirA delivered from Shigella into the vicinity of the bacterial entry site induces local degradation of the microtubules (15Yoshida S. Katayama E. Kuwae A. Mimuro H. Suzuki T. Sasakawa C. EMBO J. 2002; 21: 2923-2935Crossref PubMed Scopus (95) Google Scholar), thus resulting in the release of microtubule-associated host proteins, including GEF-H1. The release of GEF-H1 is assumed to lead to the activation of Rac1 via cross-talk with RhoA (16Yoshida S. Sasakawa C. Trends Microbiol. 2003; 11: 139-143Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 17Matsuzawa T. Kuwae A. Yoshida S. Sasakawa C. Abe A. EMBO J. 2004; 23: 3570-3582Crossref PubMed Scopus (127) Google Scholar, 18Krendel M. Zenke F.T. Bokoch G.M. Nat. Cell Biol. 2002; 4: 294-301Crossref PubMed Scopus (478) Google Scholar). IpgD possesses phosphatidylinositol phosphatase activity that catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate into phosphatidylinositol 5-monophosphate, thereby promoting local actin dynamics around the bacterial entry site (19Niebuhr K. Giuriato S. Pedron T. Philpott D.J. Gaits F. Sable J. Sheetz M.P. Parsot C. Sansonetti P.J. Payrastre B. EMBO J. 2002; 21: 5069-5078Crossref PubMed Scopus (263) Google Scholar). IpaA specifically binds the N-terminal head domain of vinculin and stimulates local actin depolymerization, which is thought to facilitate the enclosing of the macropinocytotic pockets entrapping Shigella (3Cossart P. Sansonetti P.J. Science. 2004; 304: 242-248Crossref PubMed Scopus (786) Google Scholar, 20Bourdet-Sicard R. Rüdiger M. Jockusch B.M. Gounon P. Sansonetti P.J. Tran Van Nhieu G. EMBO J. 1999; 18: 5853-5862Crossref PubMed Scopus (126) Google Scholar).Previous study indicated that IpgB1 is secreted from Shigella via the TTSS into the medium (21Buchrieser C. Glaser P. Rusniok C. Nedjari H. d'Hauteville H. Kunst F. Sansonetti P. Parsot C. Mol. Microbiol. 2000; 38: 760-771Crossref PubMed Scopus (298) Google Scholar). Since the ipgB1 gene is located upstream of the ipaBCDA operon on the large plasmid of Shigella, we speculated that IpgB1 would act as the effector protein. In this study, we have attempted to the role of IpgB1 in Shigella infection and that IpgB1 plays a major role in promoting the bacterial invasion of epithelial cells. The of HeLa cells infected with the the ipgB1 or IpgB1-hyperproducing strain that the and the scale of membrane ruffling were comparable with the level of IpgB1 production in bacteria. Since the and the of membrane ruffles in host cells by IpgB1 expression were diminished Rac1 and Cdc42 were as by treatment with inhibitor of the geranylgeranylation of Rho or by the of or by it was that IpgB1 is a novel Shigella effector as the required for promoting bacterial entry into epithelial this study, we have IpgB1 as an effector secreted from Shigella via the TTSS, which to bacterial entry into host cells. that IpgB1 expression in cells is to induce membrane ruffling via the stimulation of Rac1 and Cdc42 were on the the ipgB1 mutant significantly infection in the the cell was infected with the ipgB1 the of was significantly than that with the wild type; the or the scale of membrane ruffles in HeLa cells infected with Shigella was comparable with the level of IpgB1 production in expression of IpgB1 in HeLa cells the formation of large membrane the IpgB1-mediated formation of membrane ruffles in host cells was the or expression was with the IpgB1-mediated membrane ruffles were diminished in cells with Rho and expression of IpgB1 in cells or Shigella infection to stimulation of Rac1 and Cdc42 results that IpgB1 is a novel Shigella effector involved in promoting bacterial uptake by epithelial use trigger to enter epithelial cells (3Cossart P. Sansonetti P.J. Science. 2004; 304: 242-248Crossref PubMed Scopus (786) Google Scholar, C. Lamont R. Bacterial Invasion of Host Cells. 5. Cambridge University Press, Cambridge, UK2004: 25-57Google Scholar). bacterial activity is through the of several effectors as IpaA, IpaB, IpaC, and VirA with their host IpgB1 is to be secreted from Shigella via the in for stimulating the TTSS as by Congo to the IpgB1 secretion from Shigella can be the of Congo into the medium When we the secreted from Shigella into HeLa cells we that the IpgB1 within the cells was around the cytoplasmic membrane as well as within the membrane HeLa cells were infected with the IpgB1-hyperproducing Shigella the was by with the wild-type level the of membrane ruffles from HeLa cells infected with the IpgB1-hyperproducing Shigella was than than that with wild Thus, the results that a IpgB1 is required at an of Shigella where the IpgB1 activity in promoting entry into host of the that developed infection with the ipgB1 mutant was significantly the of that with wild than that developed infection with wild-type or ipgB1 strain that IpgB1 be involved in a in Shigella infection as the of Shigella through a cell at the of formation of membrane of the by adjacent cells, and of the that the bacterial Ohayon H. Sansonetti P.J. Parsot C. Microbiol. 1999; PubMed Scopus Google Scholar). Since the ipgB1 mutant in bacterial the formation of membrane or the ability to the host membrane, we that IpgB1 in the uptake of bacteria by adjacent cells or in required for bacterial HeLa cells expressing EGFP-IpgB1 large membrane ruffles and the EGFP-IpgB1 was associated with the membrane ruffles including the cell we various of IpgB1 with for their membrane The results that was to induce membrane the of was significantly less than that with EGFP-IpgB1 or that the N-terminal be involved in the The IpgB1 the N-terminal the or were to induce membrane ruffling in HeLa cells. the IpgB1 acid required as the TTSS P. Microbiol. Mol. Biol. 2004; PubMed Scopus Google Scholar), to be required to induce membrane ruffling. Since the acid of IpgB1 a domain to in the the membrane of IpgB1 be through the with host IpgB1-mediated membrane ruffling was in HeLa cells with GGTI-298, an inhibitor of geranylgeranylation of Rho that the IpgB1-mediated formation of membrane ruffles is on the activity of Rho as Rac1 and of on entry into host cells was M. R. M. E. A. L. J. Biol. 2003; PubMed Scopus Google Scholar, F. L. 2003; PubMed Scopus Google Scholar). that is mechanism to lead to formation of membrane ruffles in a Rho G.M. A.M. A. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, F. L. R. Nat. Cell Biol. 2002; 4: PubMed Scopus Google Scholar). example, with and the is involved in inducing formation of Rho F. L. R. Nat. Cell Biol. 2002; 4: PubMed Scopus Google Scholar). level of IpgB1-mediated formation was treatment of HeLa cells with at that a Rho formation be involved in Shigella Since the formation of large membrane ruffles by expression of EGFP-IpgB1 in HeLa cells was diminished by the Rac1 or Cdc42 into the cells, the IpgB1-mediated formation to be on the Rac1 and Cdc42 IpgB1 was expressed in cells or the cells were infected with Shigella, the of Rac1 and Cdc42 were significantly to the results of the assay. The of Rac1 and Cdc42 in cells were significantly infection with wild-type Shigella for in with that with ipgB1 mutant or the which with the study (10Burton E.A. Plattner R. Pendergast A.M. EMBO J. 2003; 22: 5471-5479Crossref PubMed Scopus (109) Google Scholar). is that the of the Rac1 activation was than that of Cdc42 the of Rac1 on formation was than that of the Cdc42 the mechanism underlying the activation of the Rho by IpgB1 to be results that IpgB1 is capable of stimulation of Rac1 and Cdc42 activities, in which IpgB1 to act on Rac1 than and (and effectors secreted via the TTSS are capable of stimulating the Rho via a with the bacterial effectors Full Text Full Text PDF PubMed Scopus Google Scholar, Nature. 1999; PubMed Scopus Google Scholar, S. A. S. M. S. Mol. Microbiol. 2000; PubMed Scopus Google Scholar). the membrane ruffles were to by Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. S. M. S. Mol. Microbiol. 2000; PubMed Scopus Google Scholar), IpgB1 acid with or IpgB1 to have to with the Rho as by the assay. a domain through which they with Rho as Rac1 and Cdc42 A. Hall A. 2002; PubMed Scopus Google Scholar), proteins, as have domain are capable of the Rho GTPases. possesses activity associated with the cell membrane via the formation of a H. M. Nature. 2003; PubMed Scopus Google Scholar, E. L. C. M. H. Nat. Cell Biol. 2002; 4: PubMed Scopus Google Scholar, J. Mol. Biol. PubMed Scopus Google Scholar). study indicated that Shigella infection of epithelial cells stimulates accumulation of the Abl tyrosine kinases around the site of bacterial which phosphorylation of an and thus leads to activation of Rac1 and Cdc42 (10Burton E.A. Plattner R. Pendergast A.M. EMBO J. 2003; 22: 5471-5479Crossref PubMed Scopus (109) Google Scholar). study indicated that which is by tyrosine with Crk and local actin polymerization involved in Shigella uptake by host cells L. Girardin S.E. Weed S.A. Karginov A.V. Olivo-Marin J.C. Parsons J.T. Sansonetti P.J. Tran Van Nhieu G. J. Cell Biol. 2004; 166: 225-235Crossref PubMed Scopus (73) Google Scholar). Since expression of IpgB1 in cells, as epithelial cell cells, cells, cells, cells, or cells, is capable of inducing membrane ruffles, we that IpgB1 in a that stimulates Rac1 and Cdc42 activities, thus promoting Shigella entry into epithelial we have to the to bacterial invasion by IpgB1 to the effectors as IpaC or it is that activity with several Shigella effectors is to induce events around the site of bacterial entry the invasiveness of HeLa cells by the ipgB1 mutant was of the level of invasiveness of the wild type (100%) as by the gentamicin protection that the in the ipgB1 mutant is to the of the and gene we that IpgB1 plays a major role in bacterial the of the membrane ruffles and the bacterial invasiveness were enhanced as the level of IpgB1 expression in epithelial cells and in the IpgB1-mediated membrane ruffles in HeLa cells were than by IpaC and VirA expression Van Nhieu G. Caron E. Hall A. Sansonetti P.J. EMBO J. 1999; 18: 3249-3262Crossref PubMed Scopus (202) Google Scholar, S. Katayama E. Kuwae A. Mimuro H. Suzuki T. Sasakawa C. EMBO J. 2002; 21: 2923-2935Crossref PubMed Scopus (95) Google Scholar). Since of the host to with IpgB1 as by a or and we that IpgB1 Rac1 and Cdc42 by with host associated with the host membrane. In of the host that with IpgB1 be to the role of IpgB1 in Shigella invasion of host cells. IntroductionShigella are highly adapted human pathogens and are the cause of bacillary dysentery. The prominent pathogenic feature of Shigella is the ability to invade a variety of cells during infection of the intestinal mucosa, including enterocytes, macrophages, dendritic cells, and neutrophils. When Shigella reach the colon, they translocate through the epithelial barrier via the M cells that overlie solitary lymphoid nodules. Once they reach the underlying M cells, Shigella invade the resident macrophages, and the infecting bacteria escape from the phagosome into the cytoplasm. Shigella multiply in the macrophage cytoplasm and finally induce cell death (1Zychlinsky A. Prevost M.C. Sansonetti P.J. Nature. 1992; 358: 167-169Crossref PubMed Scopus (775) Google Scholar, 2Suzuki T. Nakanishi K. Tsutsui H. Iwai H. Akira S. Inohara N. Chamaillard M. Nuñez G. Sasakawa C. J. Biol. Chem. 2005; 280: 14042-14050Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Shigella released from the dead macrophages enter the surrounding enterocytes through their basolateral surface by inducing large scale membrane ruffling. As soon as a bacterium is surrounded by a membrane vacuole, it immediately disrupts the vacuole membrane and escapes into the cytoplasm. Shigella multiply in the cytoplasm and move about by inducing actin polymerization at one pole of the bacterium, by which the pathogen moves within the cytoplasm as well as into the adjacent cells (3Cossart P. Sansonetti P.J. Science. 2004; 304: 242-248Crossref PubMed Scopus (786) Google Scholar). Thus, the ability of bacteria to enter the colonic epithelial cells is essential for colonization within the intestinal epithelium.For bacterial invasion of epithelial cells, Shigella (and Salmonella) use a special mechanism called the “trigger mechanism of entry,” which is characterized by macropinocytic and phagocytic events that allow cells to trap several bacterial particles simultaneously (4Sasakawa C. Lamont R. Bacterial Invasion of Host Cells. 5. Cambridge University Press, Cambridge, UK2004: 25-57Google Scholar). When Shigella comes into contact with epithelial cells, the type III secretion system (TTSS) 1The abbreviations used are: TTSS, type III secretion system; GST, glutathione S-transferase; CRIB, Cdc42/Rac-interactive binding domain; TRITC, tetramethylrhodamine isothiocyanate; FCS, fetal calf serum; CR, Congo red; PBS, phosphate-buffered saline; IPTG, isopropyl-1-thio-β-d-galactopyranoside; MOI, multiplicity of infection; GGTI, geranylgeranyltransferase inhibitor; FTI, farnesyltransferase inhibitor; siRNA, small interfering RNA; MDCK, Madin-Darby canine kidney; WT, wild type; EGFP, enhanced green fluorescent protein; LPS, lipopolysaccharide. is stimulated and delivers the effectors into the host cells and surrounding bacterial space (5Blocker A. Komoriya K. Aizawa S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 3027-3030Crossref PubMed Scopus (242) Google Scholar). The secreted effectors are capable of modulating various host functions engaged in remodeling the surface architecture of the host cell and escape from the host innate defense systems (4Sasakawa C. Lamont R. Bacterial Invasion of Host Cells. 5. Cambridge University Press, Cambridge, UK2004: 25-57Google Scholar, 6Ogawa M. Yoshimori T. Suzuki T. Sagara H. Mizushima N. Sasakawa C. Science. 2005; 307: 727-731Crossref PubMed Scopus (694) Google Scholar). Studies have indicated that several Shigella effectors, including IpaA, IpaB, IpaC, IpgD, and VirA, secreted via the TTSS are involved in stimulating the reorganization of F-actin and microtubule cytoskeletons that trigger the bacterial uptake by host cells. IpaB, for example, is capable of binding to the hyaluronic acid receptor CD44 (7Skoudy A. Mounier J. Aruffo A. Ohayon H. Gounon P. Sansonetti P. Tran Van Nhieu G. Cell Microbiol. 2000; 2: 19-33Crossref PubMed Scopus (105) Google Scholar). The IpaB-CD44 binding occurring within rafts (8van der Goot F.G. Tran Van Nhieu G. Allaoui A. Sansonetti P. Lafont F. J. Biol. Chem. 2004; 279: 47792-47798Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 9Lafont F. Tran Van Nhieu G. Hanada K. Sansonetti P. van der Goot F.G. EMBO J. 2002; 21: 4449-4457Crossref PubMed Scopus (193) Google Scholar) is capable of stimulating the cell signaling involved in promoting Shigella invasion via the accumulation of c-Src and stimulation of the phosphorylation of cortactin and Crk with the aid of the Abl tyrosine kinases (10Burton E.A. Plattner R. Pendergast A.M. EMBO J. 2003; 22: 5471-5479Crossref PubMed Scopus (109) Google Scholar, 11Tran Van Nhieu G. Enninga J. Sansonetti P. Grompone G. Curr. Opin. Microbiol. 2005; 8: 16-20Crossref PubMed Scopus (48) Google Scholar, 12Duménil G. Sansonetti P. Tran Van Nhieu G. J. Cell Sci. 2000; 113: 71-80PubMed Google Scholar, 13Bougnères L. Girardin S.E. Weed S.A. Karginov A.V. Olivo-Marin J.C. Parsons J.T. Sansonetti P.J. Tran Van Nhieu G. J. Cell Biol. 2004; 166: 225-235Crossref PubMed Scopus (73) Google Scholar). IpaC is capable of leading to the activation of Cdc42 and Rac1, since IpaC can be integrated into the host cytoplasmic membrane, which somehow leads to the recruitment of cortactin and activation of c-Src, thus promoting local actin polymerization (3Cossart P. Sansonetti P.J. Science. 2004; 304: 242-248Crossref PubMed Scopus (786) Google Scholar, 11Tran Van Nhieu G. Enninga J. Sansonetti P. Grompone G. Curr. Opin. Microbiol. 2005; 8: 16-20Crossref PubMed Scopus (48) Google Scholar, 14Tran Van Nhieu G. Caron E. Hall A. Sansonetti P.J. EMBO J. 1999; 18: 3249-3262Crossref PubMed Scopus (202) Google Scholar). VirA delivered from Shigella into the vicinity of the bacterial entry site induces local degradation of the microtubules (15Yoshida S. Katayama E. Kuwae A. Mimuro H. Suzuki T. Sasakawa C. EMBO J. 2002; 21: 2923-2935Crossref PubMed Scopus (95) Google Scholar), thus resulting in the release of microtubule-associated host proteins, including GEF-H1. The release of GEF-H1 is assumed to lead to the activation of Rac1 via cross-talk with RhoA (16Yoshida S. Sasakawa C. Trends Microbiol. 2003; 11: 139-143Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 17Matsuzawa T. Kuwae A. Yoshida S. Sasakawa C. Abe A. EMBO J. 2004; 23: 3570-3582Crossref PubMed Scopus (127) Google Scholar, 18Krendel M. Zenke F.T. Bokoch G.M. Nat. Cell Biol. 2002; 4: 294-301Crossref PubMed Scopus (478) Google Scholar). IpgD possesses phosphatidylinositol phosphatase activity that catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate into phosphatidylinositol 5-monophosphate, thereby promoting local actin dynamics around the bacterial entry site (19Niebuhr K. Giuriato S. Pedron T. Philpott D.J. Gaits F. Sable J. Sheetz M.P. Parsot C. Sansonetti P.J. Payrastre B. EMBO J. 2002; 21: 5069-5078Crossref PubMed Scopus (263) Google Scholar). IpaA specifically binds the N-terminal head domain of vinculin and stimulates local actin depolymerization, which is thought to facilitate the enclosing of the macropinocytotic pockets entrapping Shigella (3Cossart P. Sansonetti P.J. Science. 2004; 304: 242-248Crossref PubMed Scopus (786) Google Scholar, 20Bourdet-Sicard R. Rüdiger M. Jockusch B.M. Gounon P. Sansonetti P.J. Tran Van Nhieu G. EMBO J. 1999; 18: 5853-5862Crossref PubMed Scopus (126) Google Scholar).Previous study indicated that IpgB1 is secreted from Shigella via the TTSS into the medium (21Buchrieser C. Glaser P. Rusniok C. Nedjari H. d'Hauteville H. Kunst F. Sansonetti P. Parsot C. Mol. Microbiol. 2000; 38: 760-771Crossref PubMed Scopus (298) Google Scholar). Since the ipgB1 gene is located upstream of the ipaBCDA operon on the large plasmid of Shigella, we speculated that IpgB1 would act as the effector protein. In this study, we have attempted to the role of IpgB1 in Shigella infection and that IpgB1 plays a major role in promoting the bacterial invasion of epithelial cells. The of HeLa cells infected with the the ipgB1 or IpgB1-hyperproducing strain that the and the scale of membrane ruffling were comparable with the level of IpgB1 production in bacteria. Since the and the of membrane ruffles in host cells by IpgB1 expression were diminished Rac1 and Cdc42 were as by treatment with inhibitor of the geranylgeranylation of Rho or by the of or by it was that IpgB1 is a novel Shigella effector as the required for promoting bacterial entry into epithelial cells.