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• W2142072948 abstract "Salmonella Typhimurium specifically targets antigen-sampling microfold (M) cells to translocate across the gut epithelium. Although M cells represent a small proportion of the specialized follicular-associated epithelium (FAE) overlying mucosa-associated lymphoid tissues, their density increases during Salmonella infection, but the underlying molecular mechanism remains unclear. Using in vitro and in vivo infection models, we demonstrate that the S. Typhimurium type III effector protein SopB induces an epithelial-mesenchymal transition (EMT) of FAE enterocytes into M cells. This cellular transdifferentiation is a result of SopB-dependent activation of Wnt/β-catenin signaling leading to induction of both receptor activator of NF-κB ligand (RANKL) and its receptor RANK. The autocrine activation of RelB-expressing FAE enterocytes by RANKL/RANK induces the EMT-regulating transcription factor Slug that marks epithelial transdifferentiation into M cells. Thus, via the activity of a single secreted effector, S. Typhimurium transforms primed epithelial cells into M cells to promote host colonization and invasion. Salmonella Typhimurium specifically targets antigen-sampling microfold (M) cells to translocate across the gut epithelium. Although M cells represent a small proportion of the specialized follicular-associated epithelium (FAE) overlying mucosa-associated lymphoid tissues, their density increases during Salmonella infection, but the underlying molecular mechanism remains unclear. Using in vitro and in vivo infection models, we demonstrate that the S. Typhimurium type III effector protein SopB induces an epithelial-mesenchymal transition (EMT) of FAE enterocytes into M cells. This cellular transdifferentiation is a result of SopB-dependent activation of Wnt/β-catenin signaling leading to induction of both receptor activator of NF-κB ligand (RANKL) and its receptor RANK. The autocrine activation of RelB-expressing FAE enterocytes by RANKL/RANK induces the EMT-regulating transcription factor Slug that marks epithelial transdifferentiation into M cells. Thus, via the activity of a single secreted effector, S. Typhimurium transforms primed epithelial cells into M cells to promote host colonization and invasion. S. Typhimurium transforms FAE enterocytes into M cells in a RANKL-dependent manner RANKL-induced transformation requires NF-kB and Wnt/β-catenin signaling S. Typhimurium SopB is crucial for Wnt/β-catenin activation and cellular transformation S. Typhimurium effector SopB increases the number of M cells in vivo Salmonella are important bacterial pathogens that have coevolved with their hosts to modulate cellular functions to successfully survive and replicate intracellularly. In the case of Salmonella enterica serovar Typhimurium (S. Typhimurium), following adherence to epithelial cells via fimbrial adhesins, the bacterium uses the type III secretion system (TTSS)-dependent translocation of effector proteins to actively engage with regulators of the cellular cytoskeleton to induce its uptake (Galan and Zhou, 2000Galan J.E. Zhou D. Striking a balance: modulation of the actin cytoskeleton by Salmonella.Proc. Natl. Acad. Sci. USA. 2000; 97: 8754-8761Crossref PubMed Scopus (215) Google Scholar). Although S. Typhimurium has been demonstrated to invade different cell types to aid colonization and persistence in the host (Haraga et al., 2008Haraga A. Ohlson M.B. Miller S.I. Salmonellae interplay with host cells.Nat. Rev. Microbiol. 2008; 6: 53-66Crossref PubMed Scopus (597) Google Scholar), it can also traverse the epithelial barrier by preferentially entering M cells (Jones et al., 1994Jones B.D. Ghori N. Falkow S. Salmonella typhimurium initiates murine infection by penetrating and destroying the specialized epithelial M cells of the Peyer’s patches.J. Exp. Med. 1994; 180: 15-23Crossref PubMed Scopus (708) Google Scholar). M cells constitute a small subset of highly specialized follicle-associated epithelium (FAE) enterocytes overlying lymphoid follicles in the gut, and are characterized by an irregular brush border, a reduced glycocalyx and lysosomal apparatus, and a capacity to efficiently transcytose a wide variety of macromolecules and micro-organisms from the gut lumen to the underlying immune inductive Peyer’s patches (PPs) (Kraehenbuhl and Neutra, 2000Kraehenbuhl J.P. Neutra M.R. Epithelial M cells: differentiation and function.Annu. Rev. Cell Dev. Biol. 2000; 16: 301-332Crossref PubMed Scopus (395) Google Scholar). In addition to Salmonella, many other pathogenic bacteria, viruses, and prions take advantage of these unique features of M cells to gain safe passage across the intestinal barrier (Donaldson et al., 2012Donaldson D.S. Kobayashi A. Ohno H. Yagita H. Williams I.R. Mabbott N.A. M cell-depletion blocks oral prion disease pathogenesis.Mucosal Immunol. 2012; 5: 216-225Crossref PubMed Scopus (70) Google Scholar; Sansonetti and Phalipon, 1999Sansonetti P.J. Phalipon A. M cells as ports of entry for enteroinvasive pathogens: mechanisms of interaction, consequences for the disease process.Semin. Immunol. 1999; 11: 193-203Crossref PubMed Scopus (209) Google Scholar). Despite the important role of M cells in mucosal immunity, little is known about their lineage and development. Recently, receptor activator of NF-κB ligand (RANKL) was shown to be necessary and sufficient for initiation of M cell development, but the specific underlying mechanism(s) was uncertain (Knoop et al., 2009Knoop K.A. Kumar N. Butler B.R. Sakthivel S.K. Taylor R.T. Nochi T. Akiba H. Yagita H. Kiyono H. Williams I.R. RANKL is necessary and sufficient to initiate development of antigen-sampling M cells in the intestinal epithelium.J. Immunol. 2009; 183: 5738-5747Crossref PubMed Scopus (221) Google Scholar). Debate continues about whether M cells arise from lymphoid follicle-associated crypts (FACs) or ordinary crypts (OCs) and whether M cells represent a distinct lineage or derive from FAE enterocytes that have the plasticity to transition into M cells following exposure to appropriate stimuli. Certain microbes appear to exploit the innate plasticity of cells to trigger their transformation into a cell phenotype that suits their habitat, as has been demonstrated for Mycobacterium leprae in Schwann cells (Rambukkana et al., 2002Rambukkana A. Zanazzi G. Tapinos N. Salzer J.L. Contact-dependent demyelination by Mycobacterium leprae in the absence of immune cells.Science. 2002; 296: 927-931Crossref PubMed Scopus (164) Google Scholar). Intestinal epithelial cells can dramatically alter their morphology to become motile, fibroblast-like mesenchymal cells in a process referred as epithelial-mesenchymal transition (EMT). This process and the reverse, mesenchymal-epithelial transition, occur repeatedly during normal embryonic development as well as during pathological changes like tissue fibrosis or tumor metastasis (Thiery et al., 2009Thiery J.P. Acloque H. Huang R.Y. Nieto M.A. Epithelial-mesenchymal transitions in development and disease.Cell. 2009; 139: 871-890Abstract Full Text Full Text PDF PubMed Scopus (7557) Google Scholar) or chronic inflammation following certain bacterial infections (Ferreira et al., 2008Ferreira A.C. Isomoto H. Moriyama M. Fujioka T. Machado J.C. Yamaoka Y. Helicobacter and gastric malignancies.Helicobacter. 2008; 13: 28-34Crossref PubMed Scopus (46) Google Scholar). EMT is most commonly associated with loss of epithelial junction protein E-cadherin and an increase in intermediate filament protein vimentin, both of which are under control of the transcription factor Slug (Snail homolog 2; SNAI2) (Bolós et al., 2003Bolós V. Peinado H. Pérez-Moreno M.A. Fraga M.F. Esteller M. Cano A. The transcription factor Slug represses E-cadherin expression and induces epithelial to mesenchymal transitions: a comparison with Snail and E47 repressors.J. Cell Sci. 2003; 116: 499-511Crossref PubMed Scopus (925) Google Scholar). Slug gene expression is regulated by Wnt/β-catenin signaling that drives EMT during embryonic development and tumor metastasis (Vallin et al., 2001Vallin J. Thuret R. Giacomello E. Faraldo M.M. Thiery J.P. Broders F. Cloning and characterization of three Xenopus slug promoters reveal direct regulation by Lef/beta-catenin signaling.J. Biol. Chem. 2001; 276: 30350-30358Crossref PubMed Scopus (140) Google Scholar). β-catenin is typically degraded through the ubiquitin proteosome pathway that involves a complex of proteins including adenomatous polyposis coli (APC), Axin, and the glycogen synthase kinase 3β (GSK3β). Dissociation of this complex by inhibitory GSK3β phosphorylation increases the cytosolic pool of β-catenin. The free β-catenin then translocates to the cell nucleus and mediates transcriptional regulation by forming a complex with members of the T cell factor (TCF) family of transcription factors to induce EMT-specific target genes including Slug and Vimentin (Nelson and Nusse, 2004Nelson W.J. Nusse R. Convergence of Wnt, beta-catenin, and cadherin pathways.Science. 2004; 303: 1483-1487Crossref PubMed Scopus (2234) Google Scholar). Given the evidence that S. Typhimurium preferentially targets and promotes an increase in numbers of antigen-sampling M cells, we investigated the molecular mechanisms underlying this increase. We show here that S. Typhimurium activates Wnt/β-catenin signaling and events similar to EMT that transform a subset of FAC-derived epithelial cells to a cell type that phenotypically and functionally resembles M cells. We demonstrate that the S. Typhimurium TTSS effector protein SopB is necessary and sufficient to induce cellular transformation by activating Wnt/β-catenin signaling-mediated RANKL expression. We propose that S. Typhimurium facilitates mucosal penetration and host colonization by transforming these primed epithelial cells into M cells. To investigate Salmonella-mediated M cell transformation, primary epithelial cells cultured from FAC (see Figure S1 online) isolated from regions of bovine terminal rectum rich in lymphoid follicles (Mahajan et al., 2005Mahajan A. Naylor S. Mills A.D. Low J.C. Mackellar A. Hoey D.E. Currie C.G. Gally D.L. Huntley J. Smith D.G. Phenotypic and functional characterisation of follicle-associated epithelium of rectal lymphoid tissue.Cell Tissue Res. 2005; 321: 365-374Crossref PubMed Scopus (18) Google Scholar) were challenged with S. Typhimurium, and examined for changes associated with M cell development and differentiation. Prior to infection, 4%–6% of the epithelial cells in these cultures were positive for the M cell marker vimentin (Figures 1A and 1B ) (Tahoun et al., 2011Tahoun A. Siszler G. Spears K. McAteer S. Tree J. Paxton E. Gillespie T.L. Martinez-Argudo I. Jepson M.A. Shaw D.J. et al.Comparative analysis of EspF variants in inhibition of Escherichia coli phagocytosis by macrophages and inhibition of E. coli translocation through human- and bovine-derived M cells.Infect. Immun. 2011; 79: 4716-4729Crossref PubMed Scopus (22) Google Scholar). During early stages of infection (at 10 min), Salmonella typically invaded cells that were positive for vimentin (Figure 1A) and expressed sparse, short, or no microvilli or microfolds typical of M cells (Figure 1C). However, at 30 min postinfection, bacteria were also seen to interact with non-vimentin-expressing enterocytes (Figure 1A) with well-developed microvilli (Figure 1C). Over the course of the infection (180 min), the number of vimentin-positive cells (Figures 1A and 1B and Figure S2A), the number of bacteria adherent to these cells (Figure S2B), and the level of vimentin expression both at the protein (Figure 1D) and transcript levels (Figure S1J) increased, and the cells to which Salmonella bacteria adhered almost completely lost their microvilli (arrow) (Figure 1C). Moreover, the expression of E-cadherin, a typical epithelial marker, was reduced over the infection time course (Figure 1D and Figure S1J). Salmonella infection induced the expression of RANKL, which has been shown in mice to be essential for M cell development and differentiation (Knoop et al., 2009Knoop K.A. Kumar N. Butler B.R. Sakthivel S.K. Taylor R.T. Nochi T. Akiba H. Yagita H. Kiyono H. Williams I.R. RANKL is necessary and sufficient to initiate development of antigen-sampling M cells in the intestinal epithelium.J. Immunol. 2009; 183: 5738-5747Crossref PubMed Scopus (221) Google Scholar), as well as of its receptor RANK (Figure 1D and Figure S1J). The S. Typhimurium-mediated increase in number of vimentin-positive cells (Figures 1E), the enhanced expression of vimentin, its transcriptional regulator Slug, and Spi-B, the master regulator of M cell maturation and differentiation (de Lau et al., 2012de Lau W. Kujala P. Schneeberger K. Middendorp S. Li V.S. Barker N. Martens A. Hofhuis F. Dekoter R.P. Peters P.J. et al.Peyer’s Patch M cells derive from Lgr5+ stem cells require SpiB and are induced by RankL in cultured ‘organoids’.Mol. Cell. Biol. 2012; 32: 3639-3647Crossref PubMed Scopus (162) Google Scholar; Kanaya et al., 2012Kanaya T. Hase K. Takahashi D. Fukuda S. Hoshino K. Sasaki I. Hemmi H. Knoop K.A. Kumar N. Sato M. et al.The Ets transcription factor Spi-B is essential for the differentiation of intestinal microfold cells.Nat. Immunol. 2012; 13: 729-736Crossref PubMed Scopus (150) Google Scholar), were reduced in the presence of osteoprotegerin (OPG) (Figures 1F and 1G and Figure S1K), a competitive decoy receptor for RANKL (Standal et al., 2002Standal T. Seidel C. Hjertner O. Plesner T. Sanderson R.D. Waage A. Borset M. Sundan A. Osteoprotegerin is bound, internalized, and degraded by multiple myeloma cells.Blood. 2002; 100: 3002-3007Crossref PubMed Scopus (219) Google Scholar) (Figure 1E). Thus, Salmonella infection leads to an increase in number of cells positive for the M cell marker vimentin in this culture model, which is associated with an enhanced expression of the RANKL growth factor and its cellular receptor RANK. No apoptotic changes were observed over the course of S. Typhimurium infection (data not shown). Ultrastructural studies on proximal and lymphoid-dense terminal bovine rectal tissue revealed morphologically distinct crypt populations at these two sites (Figure 2A). Unlike OC, which had apical round orifice in the proximal rectum, most FAC in the terminal rectum exhibited longitudinal or oval orifice morphologically similar to “dome-associated crypts” of mouse PPs previously identified as the source for FAE enterocytes and M cell progenitors (Gebert et al., 1999Gebert A. Fassbender S. Werner K. Weissferdt A. The development of M cells in Peyer’s patches is restricted to specialized dome-associated crypts.Am. J. Pathol. 1999; 154: 1573-1582Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). We next determined whether epithelial cells cultured from crypts from these distinct regions respond differently to RANKL stimulation. Unstimulated cells derived from both FAC and OC were comparable in expression for RANK, vimentin, and epithelial-specific marker E-cadherin (Figure 2B). In contrast to RANKL-treated cells derived from OC, FAC-epithelial cells treated with RANKL showed increased levels of vimentin as well as an increase in number of cells positive for the M cell marker vimentin (Figures 2C and 2D). We next analyzed RANKL-treated cells for expression of Slug, the transcriptional regulator central to RANKL-induced EMT (Odero-Marah et al., 2008Odero-Marah V.A. Wang R. Chu G. Zayzafoon M. Xu J. Shi C. Marshall F.F. Zhau H.E. Chung L.W.K. Receptor activator of NF- B Ligand (RANKL) expression is associated with epithelial to mesenchymal transition in human prostate cancer cells.Cell Res. 2008; 18: 858-870Crossref PubMed Scopus (114) Google Scholar). A RANKL-dependent increase in Slug expression was detected in epithelial cells derived from FAC and not in those from the OC (Figure 2C). Enhanced expression of Slug and vimentin in RANKL-treated FAC-epithelial cells was also confirmed by immunofluorescence (IF) (Figure S3A). Similarly, an increase in SNAI2 (which encodes Slug), VIM (which encodes vimentin), and TNFRSF11A (which encodes RANK) transcript expression was observed (Figure S3B). These results indicate that RANKL-induced EMT is the mechanistic basis underpinning epithelial cell transformation into M cells as identified during increased levels of S. Typhimurium translocation across RANKL-treated FAC-epithelial cells (Figure 2E). As both the NF-κB and the Wnt/β-catenin signaling pathways are known to induce the EMT transcription factor Slug (Min et al., 2008Min C. Eddy S.F. Sherr D.H. Sonenshein G.E. NF-kappaB and epithelial to mesenchymal transition of cancer.J. Cell. Biochem. 2008; 104: 733-744Crossref PubMed Scopus (334) Google Scholar; Zhou et al., 2004Zhou B.P. Deng J. Xia W. Xu J. Li Y.M. Gunduz M. Hung M.C. Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition.Nat. Cell Biol. 2004; 6: 931-940Crossref PubMed Scopus (1319) Google Scholar), we examined their role in RANKL-mediated cell transformation. Epithelial cells derived from FAC were treated with RANKL in the presence of pharmacological inhibitors specific to each of these signaling pathways, and examined for the RANKL-induced expression of vimentin and β-catenin (Figure 3A and Figure S3C). In the presence of NF-κB inhibitor SN50 as well as the β-catenin inhibitor FH535, vimentin expression was suppressed (Figure 3A). In contrast, in the presence of the proteosomal inhibitor MG132, expression of vimentin and β-catenin was increased (Figure 3A and Figure S3C). To examine whether RANKL regulates SNAI2 and VIM promoter activity via the NF-κB and Wnt/β-catenin signaling pathways, luciferase reporter assays were performed in the presence of SN50 and FH535. Both inhibitors suppressed SNAI2 and VIM promoter activity in RANKL-treated epithelial cells, while GSK3β inhibitors (SB415286 and LiCl) enhanced their activity (Figure 3B). Together, these results indicate a role for both the NF-κB and Wnt/β-catenin signaling pathways in RANKL-mediated EMT (Vuoriluoto et al., 2011Vuoriluoto K. Haugen H. Kiviluoto S. Mpindi J.P. Nevo J. Gjerdrum C. Tiron C. Lorens J.B. Ivaska J. Vimentin regulates EMT induction by Slug and oncogenic H-Ras and migration by governing Axl expression in breast cancer.Oncogene. 2011; 30: 1436-1448Crossref PubMed Scopus (481) Google Scholar). In line with these findings, RANKL-mediated enhanced translocation of S. Typhimurium was significantly reduced in the presence of OPG and SN50, the RANKL and NF-κB inhibitors, respectively (Figure 3C), suggesting that NF-κB signaling plays a role in enhancing S. Typhimurium translocation across epithelial cells. In addition, RANKL treatment also enhanced bacterial uptake by vimentin-positive cells (Figure S4), suggesting a role of RANKL in functional activity of M cells. These results suggest a role for RANKL in inducing the epithelial transformation of a subset of cells derived specifically from FAC and not from the OC. We reasoned that this cellular restriction was due to differential expression of epithelial factors critical to RANKL signaling. We next compared the expression of RelB, the noncanonical NF-κB transcription factor essential for RANKL-mediated cellular differentiation (Vaira et al., 2008Vaira S. Johnson T. Hirbe A.C. Alhawagri M. Anwisye I. Sammut B. O’Neal J. Zou W. Weilbaecher K.N. Faccio R. Novack D.V. RelB is the NF-kappaB subunit downstream of NIK responsible for osteoclast differentiation.Proc. Natl. Acad. Sci. USA. 2008; 105: 3897-3902Crossref PubMed Scopus (121) Google Scholar) between cells from each site, as its expression has been shown to be restricted to certain cell types (Yilmaz et al., 2003Yilmaz Z.B. Weih D.S. Sivakumar V. Weih F. RelB is required for Peyer’s patch development: differential regulation of p52-RelB by lymphotoxin and TNF.EMBO J. 2003; 22: 121-130Crossref PubMed Scopus (179) Google Scholar). RelB expression was restricted to FAC-epithelial cells only (Figure 3D). To further analyze the role of RelB in inducing RANKL-mediated cellular transformation, RelB translation was silenced by RNA interference in FAC-epithelial cells. Epithelial cells treated with RelB siRNAs were not responsive to RANKL, demonstrated by a failure to upregulate Slug expression (Figure 3E) and S. Typhimurium translocation across the cultured cells (Figure 3F), suggesting that RelB expression restricts RANKL responsiveness to FAC-derived epithelial cells. We next examined if the S. Typhimurium-mediated increased levels of RANKL, vimentin, and Slug were a consequence of GSK3β-dependent Wnt/β-catenin signaling (Gilles et al., 2003Gilles C. Polette M. Mestdagt M. Nawrocki-Raby B. Ruggeri P. Birembaut P. Foidart J.M. Transactivation of vimentin by beta-catenin in human breast cancer cells.Cancer Res. 2003; 63: 2658-2664PubMed Google Scholar; Shin et al., 2005Shin C.S. Her S.J. Kim J.A. Kim D.H. Kim S.W. Kim S.Y. Kim H.S. Park K.H. Kim J.G. Kitazawa R. et al.Dominant negative N-cadherin inhibits osteoclast differentiation by interfering with beta-catenin regulation of RANKL, independent of cell-cell adhesion.J. Bone Miner. Res. 2005; 20: 2200-2212Crossref PubMed Scopus (24) Google Scholar; Vallin et al., 2001Vallin J. Thuret R. Giacomello E. Faraldo M.M. Thiery J.P. Broders F. Cloning and characterization of three Xenopus slug promoters reveal direct regulation by Lef/beta-catenin signaling.J. Biol. Chem. 2001; 276: 30350-30358Crossref PubMed Scopus (140) Google Scholar). Immunoblot and IF analysis showed that following S. Typhimurium infection, β-catenin was redistributed from the adherens junctions to the cytoplasm and nucleus (Figures 4A and 4B ). The increased intranuclear localization of β-catenin and Slug (Figures 4C and 4D) suggested a downstream effect of S. Typhimurium-mediated GSK3β suppression. GSK3β is a known target for PI3K/Akt kinases (Doble and Woodgett, 2003Doble B.W. Woodgett J.R. GSK-3: tricks of the trade for a multi-tasking kinase.J. Cell Sci. 2003; 116: 1175-1186Crossref PubMed Scopus (1760) Google Scholar). In addition to its role in phosphorylation of cytosolic β-catenin and other zinc-finger transcription factors like Snail, which marks these for ubiquitination and subsequent proteosomal degradation (Doble and Woodgett, 2003Doble B.W. Woodgett J.R. GSK-3: tricks of the trade for a multi-tasking kinase.J. Cell Sci. 2003; 116: 1175-1186Crossref PubMed Scopus (1760) Google Scholar; Zhou et al., 2004Zhou B.P. Deng J. Xia W. Xu J. Li Y.M. Gunduz M. Hung M.C. Dual regulation of Snail by GSK-3beta-mediated phosphorylation in control of epithelial-mesenchymal transition.Nat. Cell Biol. 2004; 6: 931-940Crossref PubMed Scopus (1319) Google Scholar), GSK3β also represses NF-κB-dependent expression of target genes associated with EMT (Bachelder et al., 2005Bachelder R.E. Yoon S.O. Franci C. de Herreros A.G. Mercurio A.M. Glycogen synthase kinase-3 is an endogenous inhibitor of Snail transcription: implications for the epithelial-mesenchymal transition.J. Cell Biol. 2005; 168: 29-33Crossref PubMed Scopus (335) Google Scholar). To confirm the role of GSK3β in S. Typhimurium-induced upregulation of β-catenin and thus activation of Wnt/β-catenin pathway, cells were infected in the presence of the following pharmacological inhibitors specific to GSK3β activation and its target substrates: LY294002 (20 μM), an inhibitor of PI3K pathway; SN50 (50 μM), an inhibitor of NF-κB pathway; Akti-1/2 (20 μM), an allosteric inhibitor of Akt1 and Akt2 isozymes; and MG132 (20 μM), a proteosome inhibitor and SB415286 (25 μM), a GSK3β inhibitor. At the concentrations used, none of these inhibitors showed any effect on viability of S. Typhimurium or epithelial cells (data not shown). As seen in Figure 4E, treatment with SN50, LY294002, or Akti-1/2 suppressed β-catenin, RANKL, Slug, and vimentin expression, while SB415286 and MG132 elevated their levels in S. Typhimurium-infected epithelial cells. Pretreatment of cells with LY294002 and Akti-1/2 completely inhibited S. Typhimurium-induced GSK3β phophorylation (Ser-9)/inactivation (Figure 4F). Together, these results suggest that S. Typhimurium first activates PI3K and consecutively Akt, which leads to inhibitory phosphorylation (Ser-9) of GSK3β. Inactivation of GSK3β leads to an increase in cytosolic β-catenin levels and induces the Wnt/β-catenin signaling pathway. To establish if Salmonella-specific secreted effector proteins are responsible for EMT, cells were exposed to viable or heat-killed (STHK) wild-type (WT) S. Typhimurium or isogenic derivative strain that lacks a functional Salmonella pathogenicity island-1 (SPI-1)-encoded TTSS. In contrast to WT S. Typhimurium, the heat-killed and SPI-1 mutant Salmonella induce much lower levels of expression of RANKL, Slug, and vimentin (Figure 5A), suggesting that SPI-1-encoded effector proteins are involved in the transformation process. We next focused on the SPI-1-encoded effector proteins SopB, SopE, and SopE2 that specifically activate Rho family GTPases, and SipA that directly interacts with actin (McGhie et al., 2001McGhie E.J. Hayward R.D. Koronakis V. Cooperation between actin-binding proteins of invasive Salmonella: SipA potentiates SipC nucleation and bundling of actin.EMBO J. 2001; 20: 2131-2139Crossref PubMed Scopus (139) Google Scholar), using mutant bacterial strains lacking expression of these proteins. In comparison to WT S. Typhimurium and the other isogenic mutants, the S. Typhimurium ΔsopB strain exhibited a reduced adherence phenotype (data not shown) as well as suppressed induction of RANKL, Slug, and vimentin expression at both protein (Figures 5B) and transcript levels (Figure S5A). Unlike WT, the ΔsopB mutant S. Typhimurium strain showed no effect on number of vimentin-positive cells in culture (Figure S5C). S. Typhimurium suppressed E-cadherin (CDH1) transcription in a Sop-B-dependent manner (Figure S5B). Immunoblot analysis of protein lysates revealed an induction of GSK3β phosphorylation (Ser-9) by the WT and other mutants except for the ΔsopB strain, which correlated with phosphorylated forms of β-catenin and Akt kinase for each of these respective strains (Figure 5C). Next, we determined whether SopB phosphatase activity was critical for the activation of Wnt/β-catenin signaling. Intestinal epithelial cells were infected with a sopB-deficient strain complemented with either catalytically active (pSopB) or inactive SopB mutant (pSopBCys462S) plasmids. The cell lysates were analyzed for total vimentin and β-catenin phosphorylation, as indicators of Wnt/β-catenin signaling. While the isogenic sopB deletion strain (ΔsopB) showed enhanced phospho-β-catenin, this was also the case for complementation with the catalytically inactive mutant, SopBCys462S (Figure 5D). However, complementation of the ΔsopB strain with a catalytically active SopB (pSopB) restored the WT phenotype. This indicates that the conserved cysteine residue Cys-462, which is essential for the inositol phosphatase activity of SopB, is also necessary for activating the Wnt/β-catenin signaling pathway. To further confirm the role of SopB in Wnt/β-catenin signaling regulation and RANKL expression, intestinal epithelial cells were cotransfected with a luciferase reporter plasmid containing WT (TOPflash) or mutated (FOPflash) LEF/TCF binding sites and a SopB expression plasmid. As shown in Figure 5E, SopB directly upregulated luciferase activity within 48 hr posttransfection, confirming a role of SopB in regulation of LEF/TCF activity. Recent studies on the regulation of RANKL (encoded by TNFSF11) expression have identified several transactivators that cooperate to activate its transcription, including NF-κB and β-catenin/TCF signaling (Shin et al., 2005Shin C.S. Her S.J. Kim J.A. Kim D.H. Kim S.W. Kim S.Y. Kim H.S. Park K.H. Kim J.G. Kitazawa R. et al.Dominant negative N-cadherin inhibits osteoclast differentiation by interfering with beta-catenin regulation of RANKL, independent of cell-cell adhesion.J. Bone Miner. Res. 2005; 20: 2200-2212Crossref PubMed Scopus (24) Google Scholar). To further confirm the role of SopB in the regulation of RANKL expression, RANKL-reporter activity was measured in the presence of pharmacological inhibitors (Figure 5F). In the presence of inhibitors for PI3K (LY294002), Akt1 and Akt2 (Akti-1/2), β-catenin/TCF (FH535), and NF-κB (SN50), the RANKL luciferase activity was significantly reduced. However, luciferase activity was significantly increased by SB415286, a GSK3β inhibitor. In summary, these data indicate that SopB-mediated PI3 kinase, NF-κB, and β-catenin signaling regulate RANKL expression at the transcriptional level. To confirm that SopB effector protein is responsible for S. Typhimurium-mediated induction of M cells in vivo, murine ligated gut loops were infected with WT or the sopB mutant (ΔsopB) S. Typhimurium strains. Glycoprotein 2 (GP2) is a specific surface marker for M cells in the mouse (Nakato et al., 2009Nakato G. Fukuda S. Hase K. Goitsuka R. Cooper M.D. Ohno H. New approach for m-cell-specific molecules screening by comprehensive transcriptome analysis.DNA Res. 2009; 16: 227-235Crossref PubMed Scopus (52) Google Scholar). Ultrastructural analysis of WT S. Typhimurium-infected PPs revealed morphogenic transition of epithelial cells expressing dense microvilli in to M-cell-like cells with characteristic apical “microfold or membranous” structures (Figures 6A–6F). To determine the changes in M cell numbers, PPs from infected mice were immunostained as whole-tissue mounts for GP2, and positive cells were quantified microscopically. The number of GP2-expressing cells was significantly increased after infection with the WT, but not after infection with the ΔsopB S. Typhimurium strain (Figure 6G). Furthermore, the immunohistological staining for Slug rev" @default.
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• W2142072948 date "2012-11-01" @default.
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• W2142072948 title "Salmonella Transforms Follicle-Associated Epithelial Cells into M Cells to Promote Intestinal Invasion" @default.
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