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• W2170671189 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 characterize the role of IpgB1 in Shigella infection and found that IpgB1 plays a major role in promoting the bacterial invasion of epithelial cells. The examination of HeLa cells infected with the wild-type, the ipgB1 mutant, or IpgB1-hyperproducing strain suggested that the invasive capacity and the scale of membrane ruffling were comparable with the level of IpgB1 production in bacteria. Since the size and the frequency of membrane ruffles in host cells induced by IpgB1 expression were diminished when Rac1 and Cdc42 activities were inhibited, such as by treatment with GGTI-298 (an inhibitor of the geranylgeranylation of Rho GTPases) or by the knocking down of rac1 or cdc42 by siRNAs, it was likely that IpgB1 is a novel Shigella effector acting as the invasin required for promoting bacterial entry into epithelial cells.DISCUSSIONIn this study, we have identified IpgB1 protein as an effector secreted from Shigella via the TTSS, which acts to promote bacterial entry into host cells. We also found that IpgB1 expression in mammalian cells is able to induce membrane ruffling via the stimulation of Rac1 and Cdc42 activities. Our conclusions were based on the following results: (i) the ipgB1 mutant partially but significantly attenuated pulmonary infection in the murine model; (ii) when the MDCK cell monolayer was infected with the ipgB1 mutant, the number of plaques was significantly lower than that with the wild type; (iii) the invasive efficiency or the scale of membrane ruffles in HeLa cells infected with Shigella was comparable with the level of IpgB1 production in bacteria; (iv) ectopic expression of IpgB1 in HeLa cells induced the formation of large membrane ruffles; (v) the IpgB1-mediated formation of membrane ruffles in host cells was inhibited when the rac1 or cdc42 expression was knocked down with siRNA; (vi) the IpgB1-mediated membrane ruffles were diminished in cells treated with GGTI-298 (a Rho GTPase inhibitor); and (vii) ectopic expression of IpgB1 in cells or Shigella infection led to stimulation of Rac1 and Cdc42 activities. These results suggest that IpgB1 is a novel Shigella effector involved in promoting bacterial uptake by epithelial cells.Shigella use trigger mechanisms to enter epithelial cells (3Cossart P. Sansonetti P.J. Science. 2004; 304: 242-248Crossref PubMed Scopus (786) Google Scholar, 4Sasakawa C. Lamont R. Bacterial Invasion of Host Cells. 5. Cambridge University Press, Cambridge, UK2004: 25-57Google Scholar). This bacterial activity is gained through the interaction of several effectors such as IpaA, IpaB, IpaC, and VirA with their host target molecules (see Introduction). IpgB1 is shown to be rapidly secreted from Shigella via the TTSS. Indeed, under in vitro conditions for stimulating the TTSS such as by adding Congo red to the medium, IpgB1 secretion from Shigella can be detected only 15 min after the addition of Congo red into the medium (this study). When we visualized the IpgB1-Myc protein secreted from Shigella into HeLa cells using immunofluorescence microscopy, we observed that the IpgB1 signal within the cells was localized around the cytoplasmic membrane as well as within the membrane ruffles. Furthermore, when HeLa cells were infected with the IpgB1-hyperproducing Shigella (WT/pTB-IpgB1-Spa15), the invasive efficiency was dramatically increased by ~30-fold compared with the wild-type level (see Fig. 3E). Consistently, the size of membrane ruffles extending from HeLa cells infected with the IpgB1-hyperproducing Shigella was also more than 2 times larger than that with wild type. Thus, the results suggest that a functional IpgB1 is required at an early stage of Shigella infection, where the IpgB1 activity engages in promoting entry into host cells.The diameter of the plaques that had developed 3 days after infection with the ipgB1 mutant was significantly smaller (one-third the diameter of that with wild type) than that developed after infection with wild-type or ipgB1 complement strain (see Fig. 3B), suggesting that IpgB1 function may also be involved in a later step in Shigella infection such as the cell-cell dissemination step. Dissemination of Shigella through a cell monolayer involves at least four distinctive steps: the actin-based movement of intracellular bacteria, formation of membrane protrusions, engulfment of the protrusions by adjacent cells, and lysis of the two cellular membranes that surround the bacterial protrusion (39Page A.L. Ohayon H. Sansonetti P.J. Parsot C. Cell. Microbiol. 1999; 1: 183-193Crossref PubMed Scopus (92) Google Scholar). Since the ipgB1 mutant showed no defects in bacterial motility, the formation of membrane protrusions, or the ability to disrupt the host plasma membrane, 3K. Ohya, unpublished data. we speculate that IpgB1 also takes part in the uptake of bacteria by adjacent cells or in some undefined function required for bacterial cell-cell spreading.Since HeLa cells ectopically expressing EGFP-IpgB1 formed large membrane ruffles and the EGFP-IpgB1 signal was predominately associated with the membrane ruffles including the cell periphery, we investigated various truncated versions of IpgB1 fused with EGFP for their membrane association. The results showed that although EGFP-IpgB1-(53-208) was still able to induce membrane ruffling, the degree of association was significantly less than that with EGFP-IpgB1 (full-length IpgB1) or EGFP-IpgB1-(29-208), implying that the N-terminal 52 amino acids may be involved in the association (see Fig. 5B). The other truncated IpgB1 versions (i.e. those lacking the N-terminal 104 amino acids (EGFP-IpgB1-(105-208)), the C-terminal 55 amino acids (EGFP-IpgB1-(1-153)), or both terminal portions (EGFP-IpgB1-(53-153)) were yet unable to induce membrane ruffling in HeLa cells. Therefore, the entire IpgB1 sequence, except amino acid residues 1-29 perhaps required as the TTSS translocating signal (40Ghosh P. Microbiol. Mol. Biol. Rev. 2004; 68: 771-795Crossref PubMed Scopus (325) Google Scholar), appears to be required to induce membrane ruffling. Since the amino acid sequence of IpgB1 contains neither a predictable transmembrane domain nor significant similarity to other known proteins in the data base, the membrane association of IpgB1 may be achieved through the interaction with some host protein(s).The IpgB1-mediated membrane ruffling was strongly inhibited in HeLa cells treated with GGTI-298, an inhibitor of geranylgeranylation of Rho GTPases (see Fig. 7B), suggesting that the IpgB1-mediated formation of membrane ruffles is dependent on the activity of Rho GTPases such as Rac1 and Cdc42. A similar inhibitory effect of GGTI-298 on Salmonella entry into host cells was reported (41Forsberg M. Blomgran R. Lerm M. Särndahl E. Sebti S.M. Hamilton A. Stendahl O. Zheng L. J. Leukocyte Biol. 2003; 74: 620-629Crossref PubMed Scopus (38) Google Scholar, 42Tafazoli F. Magnusson K.E. Zheng L. Infect. Immun. 2003; 71: 872-881Crossref PubMed Scopus (64) Google Scholar). Note that there is another mechanism to lead to formation of membrane ruffles in a Rho GTPase-independent manner (43Bokoch G.M. Reilly A.M. Daniels R.H. King C.C. Olivera A. Spiegel S. Knaus U.G. J. Biol. Chem. 1998; 273: 8137-8144Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 44Vadlamudi R.K. Li F. Adam L. Nguyen D. Ohta Y. Stossel T.P. Kumar R. Nat. Cell Biol. 2002; 4: 681-690Crossref PubMed Scopus (259) Google Scholar). For example, p21-activated kinase 1 directly interacts with filamin FLNa, and the p21-activated kinase 1-FLNa interaction is involved in inducing ruffle formation independently of Rho GTPases (44Vadlamudi R.K. Li F. Adam L. Nguyen D. Ohta Y. Stossel T.P. Kumar R. Nat. Cell Biol. 2002; 4: 681-690Crossref PubMed Scopus (259) Google Scholar). Indeed, some low level of IpgB1-mediated ruffle formation was still detected upon treatment of HeLa cells with GGTI-298 at 5 μm (Fig. 7B), suggesting that a Rho GTPase-independent ruffle formation may also be partly involved in Shigella invasion. Since the formation of large membrane ruffles induced by expression of EGFP-IpgB1 in HeLa cells was greatly diminished by introducing the Rac1 or Cdc42 siRNAs into the cells, the IpgB1-mediated ruffle formation appears to mostly be dependent on the Rac1 and Cdc42 activities. Indeed, when IpgB1 was expressed in NIH3T3 cells or the cells were infected with Shigella, the levels of activated Rac1 and Cdc42 were significantly increased according to the results of the CRIB-binding assay. The levels of activated Rac1 and Cdc42 in NIH3T3 cells were significantly increased after infection with wild-type Shigella for 15 min in comparison with that with ipgB1 mutant or the uninfected control, which also agreed with the previous study (10Burton E.A. Plattner R. Pendergast A.M. EMBO J. 2003; 22: 5471-5479Crossref PubMed Scopus (109) Google Scholar). It is noteworthy that the enhancement of the IpgB1-dependent Rac1 activation was greater than that of Cdc42 (see Fig. 8). Furthermore, the inhibitory effect of Rac1 siRNA on IpgB1-induced ruffle formation was more striking than that of the Cdc42 siRNA (see Fig. 6). Although the mechanism underlying the activation of the Rho GTPases by IpgB1 remains to be elucidated, our results suggest that IpgB1 is capable of mediating stimulation of both Rac1 and Cdc42 activities, in which IpgB1 appears to act on Rac1 more efficiently than Cdc42. Salmonella, SptP (possessing GAP activity), and SopE (and SopE2) effectors (possessing GEF activity) secreted via the TTSS are capable of directly stimulating the Rho GTPases via a direct interaction with the bacterial effectors (45Hardt W.D. Chen L.M. Schuebel K.E. Bustelo X.R. Galán J.E. Cell. 1998; 93: 815-826Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar, 46Fu Y. Galán J.E. Nature. 1999; 401: 293-297Crossref PubMed Scopus (445) Google Scholar, 47Stender S. Friebel A. Linder S. Rohde M. Mirold S. Hardt W.D. Mol. Microbiol. 2000; 36: 1206-1221Crossref PubMed Scopus (232) Google Scholar). Interestingly, although the IpgB1-induced membrane ruffles were apparently similar to those induced by Salmonella SopE (45Hardt W.D. Chen L.M. Schuebel K.E. Bustelo X.R. Galán J.E. Cell. 1998; 93: 815-826Abstract Full Text Full Text PDF PubMed Scopus (658) Google Scholar, 47Stender S. Friebel A. Linder S. Rohde M. Mirold S. Hardt W.D. Mol. Microbiol. 2000; 36: 1206-1221Crossref PubMed Scopus (232) Google Scholar), IpgB1 shares no significant amino acid homology with SopE or SptP. IpgB1 does not seem to have any capacity to directly interact with the Rho GTPases as examined by the GST pull-down assay. 3K. Ohya, unpublished data. Many GEF proteins possess a Dbl homology domain through which they directly interact with Rho GTPases, such as Rac1 and Cdc42 (48Schmidt A. Hall A. Genes Dev. 2002; 16: 1587-1609Crossref PubMed Scopus (974) Google Scholar), whereas other GEF proteins, such as Dock180, have no Dbl homology domain but are still capable of activating the Rho GTPases. Dock180 possesses GEF activity associated with the cell membrane via the formation of a Dock180-RhoG-Elmo complex (49Katoh H. Negishi M. Nature. 2003; 424: 461-464Crossref PubMed Scopus (291) Google Scholar, 50Brugnera E. Haney L. Grimsley C. Lu M. Walk S.F. Tosello-Trampont A.C. Macara I.G. Madhani H. Fink G.R. Ravichandran K.S. Nat. Cell Biol. 2002; 4: 574-582Crossref PubMed Scopus (468) Google Scholar, 51Casadaban M.J. Cohen S.N. J. Mol. Biol. 1980; 138: 179-207Crossref PubMed Scopus (1739) Google Scholar). A recent study has indicated that Shigella infection of epithelial cells stimulates accumulation of the Abl tyrosine kinases around the site of bacterial entry, which increases phosphorylation of Crk, an adaptor protein, and thus eventually 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). A more recent study has indicated that cortactin, which is phosphorylated by Src tyrosine kinase, interacts with Crk and promotes local actin polymerization involved in Shigella uptake by host cells (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). Since expression of IpgB1 in mammalian cells, such as AGS (a gastric epithelial cell line) cells, MDCK cells, Caco-2 cells, COS-7 cells, or NIH3T3 cells, is also capable of inducing membrane ruffles, 3K. Ohya, unpublished data. we assume that IpgB1 may take part in a signal cascade that stimulates Rac1 and Cdc42 activities, thus promoting Shigella entry into epithelial cells.At present, we have no available data to directly evaluate the relative contribution to bacterial invasion by IpgB1 to the other known effectors such as IpaC or VirA. However, it is most likely that synergistic activity with several Shigella effectors is needed to induce profound macropynocytic events around the site of bacterial entry (see Introduction). Indeed, the invasiveness of HeLa cells by the ipgB1 mutant was ~50% of the level of invasiveness of the wild type (100%) as examined by the gentamicin protection assay (see Fig. 3E), implying that the remaining invasive capacity left in the ipgB1 mutant is attributed to the presence of the ipaC and virA gene functions. Nevertheless, we believe that IpgB1 plays a major role in bacterial entry, because the sizes of the membrane ruffles and the bacterial invasiveness were greatly enhanced as the level of IpgB1 expression increased in epithelial cells and in bacteria, respectively. Of importance, the IpgB1-mediated membrane ruffles in HeLa cells were apparently larger than those induced by IpaC and VirA expression (14Tran Van Nhieu G. Caron E. Hall A. Sansonetti P.J. EMBO J. 1999; 18: 3249-3262Crossref PubMed Scopus (202) Google Scholar, 15Yoshida S. Katayama E. Kuwae A. Mimuro H. Suzuki T. Sasakawa C. EMBO J. 2002; 21: 2923-2935Crossref PubMed Scopus (95) Google Scholar). Since none of the above host proteins has so far been found to directly interact with IpgB1 as examined by a yeast two-hybrid or GST pull-down assay, 4K. Ohya and Y. Handa, unpublished data. we speculate that IpgB1 may stimulate Rac1 and Cdc42 by interacting with some host protein(s) associated with the host membrane. In any event, identification of the host protein(s) that interacts with IpgB1 will be necessary to understand the precise 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 characterize the role of IpgB1 in Shigella infection and found that IpgB1 plays a major role in promoting the bacterial invasion of epithelial cells. The examination of HeLa cells infected with the wild-type, the ipgB1 mutant, or IpgB1-hyperproducing strain suggested that the invasive capacity and the scale of membrane ruffling were comparable with the level of IpgB1 production in bacteria. Since the size and the frequency of membrane ruffles in host cells induced by IpgB1 expression were diminished when Rac1 and Cdc42 activities were inhibited, such as by treatment with GGTI-298 (an inhibitor of the geranylgeranylation of Rho GTPases) or by the knocking down of rac1 or cdc42 by siRNAs, it was likely that IpgB1 is a novel Shigella effector acting as the invasin required for promoting bacterial entry into epithelial cells." @default.
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