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• W2014404744 abstract "The virulence factor SpvB is a crucial component for the intracellular growth and infection process of Salmonella enterica. The SpvB protein mediates the ADP-ribosylation of actin in infected cells and is assumed to be delivered directly from the engulfed bacteria into the host cell cytosol. Here we used the binary Clostridium botulinum C2 toxin as a transport system for the catalytic domain of SpvB (C/SpvB) into the host cell cytosol. A recombinant fusion toxin composed of the enzymatically inactive N-terminal domain of C. botulinum C2 toxin (C2IN) and C/SpvB was cloned, expressed, and characterized in vitro and in intact cells. When added together with C2II, the C2IN-C/SpvB fusion toxin was efficiently delivered into the host cell cytosol and ADP-ribosylated actin in various cell lines. The cellular uptake of the fusion toxin requires translocation from acidic endosomes into the cytosol and is facilitated by Hsp90. The N- and C-terminal domains of SpvB are linked by 7 proline residues. To elucidate the function of this proline region, fusion toxins containing none, 5, 7, and 9 proline residues were constructed and analyzed. The existence of the proline residues was essential for the translocation of the fusion toxins into host cell cytosol and thereby determined their cytopathic efficiency. No differences concerning the mode of action of the C2IN-C/SpvB fusion toxin and the C2 toxin were obvious as both toxins induced depolymerization of actin filaments, resulting in cell rounding. The acute cellular responses following ADP-ribosylation of actin did not immediately induce cell death of J774.A1 macrophage-like cells. The virulence factor SpvB is a crucial component for the intracellular growth and infection process of Salmonella enterica. The SpvB protein mediates the ADP-ribosylation of actin in infected cells and is assumed to be delivered directly from the engulfed bacteria into the host cell cytosol. Here we used the binary Clostridium botulinum C2 toxin as a transport system for the catalytic domain of SpvB (C/SpvB) into the host cell cytosol. A recombinant fusion toxin composed of the enzymatically inactive N-terminal domain of C. botulinum C2 toxin (C2IN) and C/SpvB was cloned, expressed, and characterized in vitro and in intact cells. When added together with C2II, the C2IN-C/SpvB fusion toxin was efficiently delivered into the host cell cytosol and ADP-ribosylated actin in various cell lines. The cellular uptake of the fusion toxin requires translocation from acidic endosomes into the cytosol and is facilitated by Hsp90. The N- and C-terminal domains of SpvB are linked by 7 proline residues. To elucidate the function of this proline region, fusion toxins containing none, 5, 7, and 9 proline residues were constructed and analyzed. The existence of the proline residues was essential for the translocation of the fusion toxins into host cell cytosol and thereby determined their cytopathic efficiency. No differences concerning the mode of action of the C2IN-C/SpvB fusion toxin and the C2 toxin were obvious as both toxins induced depolymerization of actin filaments, resulting in cell rounding. The acute cellular responses following ADP-ribosylation of actin did not immediately induce cell death of J774.A1 macrophage-like cells. Several pathogenic bacteria produce toxins and effector proteins, which attack the cytoskeleton of eukaryotic cells by mono-ADP-ribosylation of actin. In past years, we focused on the mode of action of the Clostridium botulinum C2 toxin as the prototype of binary actin-ADP-ribosylating toxins (1Aktories K. Barth H. Int. J. Med. Microbiol. 2004; 293: 557-564Crossref PubMed Scopus (20) Google Scholar). The enzyme component of the C2 toxin (C2I) 3The abbreviations used are: C2I, enzyme component of C. botulinum C2 toxin; C2IN, N-terminal domain of C2I; C2IN-C/SpvB, fusion toxin consisting of the N-terminal domain of C2I and the C-terminal domain of S. enterica SpvB; SCV, Salmonella-containing vesicles; spv, Salmonella plasmid of virulence; C/SpvB, enzyme domain of S. enterica SpvB; PBS, phosphate-buffered saline; TTSS, type III secretion system; DMEM, Dulbecco's modified Eagle's medium; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; GST, glutathione S-transferase.3The abbreviations used are: C2I, enzyme component of C. botulinum C2 toxin; C2IN, N-terminal domain of C2I; C2IN-C/SpvB, fusion toxin consisting of the N-terminal domain of C2I and the C-terminal domain of S. enterica SpvB; SCV, Salmonella-containing vesicles; spv, Salmonella plasmid of virulence; C/SpvB, enzyme domain of S. enterica SpvB; PBS, phosphate-buffered saline; TTSS, type III secretion system; DMEM, Dulbecco's modified Eagle's medium; MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt; GST, glutathione S-transferase. ADP-ribosylates G-actin at arginine 177 (2Aktories K. Bärmann M. Ohishi I. Tsuyama S. Jakobs K.H. Habermann E. Nature. 1986; 322: 390-392Crossref PubMed Scopus (384) Google Scholar). This leads to depolymerization of actin filaments and finally to cell rounding. The proteolytically activated binding/translocation component (C2IIa) forms heptamers, which assemble with C2I and bind to the cellular receptor (3Barth H. Blöcker D. Behlke J. Bergsma-Schutter W. Brisson A. Benz R. Aktories K. J. Biol. Chem. 2000; 275: 18704-18711Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). Following receptor-mediated endocytosis, C2IIa forms pores in the membrane of acidic endosomes. For translocation of the C2I protein through the lumen of these pores, a partial unfolding of C2I is required (4Haug G. Wilde C. Leemhuis J. Meyer D.K. Aktories K. Barth H. Biochemistry. 2003; 42: 15284-15291Crossref PubMed Scopus (47) Google Scholar). The subsequent refolding of C2I in the cytosol is facilitated by the host cell chaperone Hsp90 (5Haug G. Leemhuis J. Tiemann D. Meyer D.K. Aktories K. Barth H. J. Biol. Chem. 2003; 278: 32266-32274Abstract Full Text Full Text PDF PubMed Scopus (111) Google Scholar). The interaction of C2I with C2IIa is mediated by the N-terminal domain of the C2I protein (C2IN, amino acid residues 1–225). C2IN, which is enzymatically inactive and does not induce cell rounding when applied in combination with C2IIa to cells, was successfully used as an adaptor for the C2IIa-mediated delivery of different proteins into the cytosol of eukaryotic cells (6Barth H. Hofmann F. Olenik C. Just I. Aktories K. Infect. Immun. 1998; 66: 1364-1369Crossref PubMed Google Scholar).The SpvB protein from Salmonella enterica was identified as a new member of bacterial actin-ADP-ribosylating enzymes (7Otto H. Tezcan-Merdol D. Girisch R. Haag F. Rhen M. Koch-Nolte F. Mol. Microbiol. 2000; 37: 1106-1115Crossref PubMed Scopus (78) Google Scholar). We identified arginine 177 as the modification site for C/SpvB within the actin homolog Act88F (Drosophila indirect flight muscle actin) (8Hochmann H. Pust S. von Figura G. Aktories K. Barth H. Biochemistry. 2006; 45: 1271-1277Crossref PubMed Scopus (43) Google Scholar). Our finding was confirmed by a recent publication (9Margarit S.M. Davidson W. Frego L. Stebbins C.E. Structure (Lond.). 2006; 14: 1219-1229Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar) reporting that arginine 177 is the modification side in mammalian actin for SpvB. S. enterica is a Gram-negative facultative intracellular pathogen, which causes diseases ranging from mild gastroenteritis to severe systemic infections in humans (10Fierer J. Guiney D.G. J. Clin. Investig. 2001; 107: 775-780Crossref PubMed Scopus (163) Google Scholar, 11Rodriguez M. de Diego I. Mendoza M.C. J. Clin. Microbiol. 1998; 36: 3291-3296Crossref PubMed Google Scholar). During infection Salmonella grows and replicates inside macrophages in a special membrane compartment, called the Salmonella-containing vacuole (SCV) (12Guiney D.G. Lesnick M. Clin. Immunol. 2005; 114: 248-255Crossref PubMed Scopus (66) Google Scholar).As the final step in the infection process, Salmonella induces cell death of infected macrophages. Intracellular pathogenesis depends on the presence of the Salmonella pathogenicity island 2 (SPI2), which encodes for several effectors and for a type III secretion system (SPI2-encoded TTSS, for review see 13Hensel M. Shea J.E. Waterman S.R. Mundy R. Nikolaus T. Banks G. Vazquez-Torres A. Gleeson C. Fang F.C. Holden D.W. Mol. Microbiol. 1998; 30: 163-174Crossref PubMed Scopus (490) Google Scholar). A plasmid, which contains the Salmonella plasmid virulence (spv) gene cluster, is also essential for intracellular growth of Salmonella and therefore for the virulence (14Gulig P.A. Doyle T.J. Hughes J.A. Matsui H. Infect. Immun. 1998; 66: 2471-2485Crossref PubMed Google Scholar, 15Libby S.J. Adams L.G. Ficht T.A. Allen C. Whitford H.A. Buchmeier N.A. Bossie S. Guiney D.G. Infect. Immun. 1997; 65: 1786-1792Crossref PubMed Google Scholar). This plasmid comprises the four-gene operon spvABCD (16Fang F.C. Krause M. Roudier C. Fierer J. Guiney D.G. J. Bacteriol. 1991; 173: 6783-6789Crossref PubMed Google Scholar, 17Grob P. Guiney D.G. J. Bacteriol. 1996; 178: 1813-1820Crossref PubMed Google Scholar, 18Guiney D.G. Libby S. Fang F.C. Krause M. Fierer J. Trends Microbiol. 1995; 3: 275-279Abstract Full Text PDF PubMed Scopus (58) Google Scholar), in which spvB encodes for the ADP-ribosyltransferase (7Otto H. Tezcan-Merdol D. Girisch R. Haag F. Rhen M. Koch-Nolte F. Mol. Microbiol. 2000; 37: 1106-1115Crossref PubMed Scopus (78) Google Scholar, 19Tezcan-Merdol D. Nyman T. Lindberg U. Haag F. Koch-Nolte F. Rhen M. Mol. Microbiol. 2001; 39: 606-619Crossref PubMed Scopus (97) Google Scholar). It is supposed that SpvB is delivered into the cytoplasm via the SPI2-encoded TTSS (13Hensel M. Shea J.E. Waterman S.R. Mundy R. Nikolaus T. Banks G. Vazquez-Torres A. Gleeson C. Fang F.C. Holden D.W. Mol. Microbiol. 1998; 30: 163-174Crossref PubMed Scopus (490) Google Scholar, 20Ochman H. Soncini F.C. Solomon F. Groisman E.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 7800-7804Crossref PubMed Scopus (524) Google Scholar). However, the mechanism underlying the translocation of the SpvB protein across the membrane of the Salmonella-containing vacuole is not known. The N-terminal domain of SpvB might be involved in translocation of SpvB, and the C-terminal catalytic domain (C/SpvB, amino acid residues 375–591) contains the ADP-ribosyltransferase activity (7Otto H. Tezcan-Merdol D. Girisch R. Haag F. Rhen M. Koch-Nolte F. Mol. Microbiol. 2000; 37: 1106-1115Crossref PubMed Scopus (78) Google Scholar, 8Hochmann H. Pust S. von Figura G. Aktories K. Barth H. Biochemistry. 2006; 45: 1271-1277Crossref PubMed Scopus (43) Google Scholar). Between both domains a region of seven proline residues with a yet unknown function is present.Biochemical studies on the mode of action of SpvB in intact cells are limited by the fact that SpvB is not taken up when applied to cultured cells. To characterize the cellular consequences of actin-ADP-ribosylation by C/SpvB, we used the C. botulinum C2 toxin as a delivery system for the catalytic domain of SpvB into the cytosol. We constructed a recombinant fusion toxin, consisting of the N-terminal adaptor domain of the C2I component (C2IN) and the catalytic domain of SpvB (C/SpvB), and analyzed the cellular effects caused by the fusion toxin. Finally, we identified the functional role of the polyproline stretch, located between the domains C2IN and C/SpvB in the fusion toxin.EXPERIMENTAL PROCEDURESMaterials—Oligonucleotides were obtained from Hermann GbR (Freiburg, Germany). PCRs were performed with a T1 thermocycler from Biometra (Göttingen, Germany). DNA sequencing was done with an ABI PRISM 310 genetic analyzer from PerkinElmer Life Sciences. Taq polymerase was purchased from Roche Diagnostics, and Turbo Pfu polymerase was purchased from Stratagene (La Jolla, CA). Glutathione-Sepharose 4B was from Amersham Biosciences; cell culture medium was from Biochrom (Berlin, Germany); fetal calf serum was from PAN Systems (Aidenbach, Germany); and thrombin was from Sigma. ECL, Complete protease inhibitor mixture, annexin-V-FLUOS, trypsin, and trypsin inhibitor were from Roche Diagnostics. Alexa594/phalloidin, 4,6-diamidino-2-phenylindole, and ProLong Gold antifade reagent were obtained from Molecular Probes (Eugene, OR); β/γ-actin was from Cytoskeleton (Denver, CO). The 6-biotin-17-NAD was purchased from R&D Systems. The horseradish peroxidase-coupled anti-rabbit antibody was from Santa Cruz Biotechnology. The antiserum raised against C2IN was from a rabbit. The CellTiter 96® AQueous Non-radioactive Cell Proliferation Assay was from Promega (Mannheim, Germany).Cloning of C2IN-C/SpvB Fusion Toxins—The pET32a plasmid encoding the thioredoxin-S-tag-SpvB protein, encompassing residues 374–591, was kindly provided from Dr. F. Koch-Nolte (Hamburg, Germany). For cloning of an SpvB construct containing the residues 374–591, the pET encoding SpvB was amplified by PCR with 100 ng of plasmid DNA in a total volume of 50 μl with 2.5 units of TaqDNA polymerase in a reaction mixture (10 nm Tris, 15 mm MgCl2, 50 mm KCl (pH 8.3)), including deoxynucleoside triphosphates (2.5 mm each) and 12.5 pmol of the primers 5′CSpvB-BglII (5′-GGAGATCTATGATGGGAGGTAATTCATCTCG-3′) and 3′CSpvB-BglII (5′-GGAGATCTCTATGAGTTGAGTACCCTCATG-3′). The amplification was done by 25 cycles of denaturing at 94 °C for 30 s, primer annealing at 50 °C for 1 min, and extension at 72 °C for 90 s. The received PCR product was cloned into TOPO-TA vector (Invitrogen), according to the manufacturer's instructions. For fusion to the C2IN fragment, the SpvB gene was excised with BglII and cloned into the BamHI-digested pGEX-C2IN vector (6Barth H. Hofmann F. Olenik C. Just I. Aktories K. Infect. Immun. 1998; 66: 1364-1369Crossref PubMed Google Scholar). The resulting C2IN-C/SpvBΔ Pro fragment was ligated and transformed into competent Epicurean XL cells from Stratagene (La Jolla, CA). For insertion of a proline linker (according to residues 367–373 in full-length SpvB), linking C2IN to C/SpvB, a QuikChange site-directed mutagenesis, according to the QuikChange manual, was performed by using 5′C2IN(pro5)C/SpvB primer (5′-G CTT GAT TTT TAT AAT AAA GGA TCT CCG CCA CCG CCT CCT ATG ATG GGA GGT AAT TCA TCT CG-3′) and 3′C2IN(pro5)C/SpvB (5′-CG AGA TGA ATT ACC TCC CAT CAT AGG AGG CGG TGG CGG AGA TCC TTT ATT ATA AAA ATC AAG C-3′) for C2IN-C/SpvB(pro5), 5′C2IN(pro7)C/SpvB primer (5′-G CTT GAT TTT TAT AAT AAA GGA TCT CCA CCG CCA CCG CCT CCT CCG ATG ATG GGA GGT AAT TCA TCT CG-3′) and 3′C2IN(pro7)C/SpvB primer (5′-CG AGA TGA ATT ACC TCC CAT CAT CGG AGG AGG CGGTGG CGG TGG AGA TCC TTT ATT ATA AAA ATC AAG C-3′) for C2IN-C/SpvB(pro7). For the linker with 9 proline residues, a QuikChange mutagenesis of the C2IN(pro7)C/SpvB was performed by using 5′C2IN(pro9)CSpvB (5′-G CTT GAT TTT TAT AAT AAA GGA TCT CCA CCTCCA CCG CCA CCG CCT CCT CGG-3′) and 3′C2IN(pro9)CSpvB (5′-CCG AGG AGG CGG TGG CGG TGG AGG TGG AGA TCC TTT ATT ATA AAA ATC AAG C-3′). The resulting plasmids were transformed into Escherichia coli TG1 cells.Expression and Purification of Recombinant Proteins—The components of C. botulinum C2 toxin, C2I and C2II, were expressed as GST fusion proteins in E. coli BL21 cells. Proteins were purified as described previously (3Barth H. Blöcker D. Behlke J. Bergsma-Schutter W. Brisson A. Benz R. Aktories K. J. Biol. Chem. 2000; 275: 18704-18711Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar, 6Barth H. Hofmann F. Olenik C. Just I. Aktories K. Infect. Immun. 1998; 66: 1364-1369Crossref PubMed Google Scholar, 21Barth H. Roebling R. Fritz M. Aktories K. J. Biol. Chem. 2002; 277: 5074-5081Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 22Blöcker D. Behlke J. Aktories K. Barth H. Infect. Immun. 2001; 69: 2980-2987Crossref PubMed Scopus (78) Google Scholar) and incubated with thrombin (3.25 NIH units/ml of bead suspension) for cleavage of the GST domain. C2II was activated with trypsin for 30 min at 37 °C (3Barth H. Blöcker D. Behlke J. Bergsma-Schutter W. Brisson A. Benz R. Aktories K. J. Biol. Chem. 2000; 275: 18704-18711Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar). The C/SpvB protein was purified following the protocol of Otto et al. (7Otto H. Tezcan-Merdol D. Girisch R. Haag F. Rhen M. Koch-Nolte F. Mol. Microbiol. 2000; 37: 1106-1115Crossref PubMed Scopus (78) Google Scholar). However, after isopropyl 1-thio-β-d-galactopyranoside induction the E. coli cells grew at 37 °C, and no sterile filtration was done. The proteins C2IN-C/SpvB(pro5), C2IN-C/SpvB(pro7), C2IN-C/SpvB(pro9), and C2IN-C/SpvB(Δpro) were expressed as recombinant glutathione S-transferase fusion proteins in E. coli. Bacteria were grown at 37 °C in 2 liters of LB medium, containing 100 μg/ml ampicillin to an absorbance (600 nm) of 0.8. After addition of isopropyl 1-thio-β-d-galactopyranoside the cultures were grown for 18 h at 29 °C. The bacteria were sedimented at 5.000 × g (10 min, 4 °C) and resuspended in ice-cold lysis buffer (10 mm NaCl, 20 mm Tris-HCl (pH 7.4), 1% Triton X-100, and 1 mm phenylmethylsulfonyl fluoride). Following sonification, the bacterial debris was sedimented at 12.000 × g (10 min, 4 °C), resuspended, and sedimented again. The supernatant was added to a 50% slurry of glutathione-Sepharose 4B in PBS (400 μl/1 liter) and incubated for 60 min at room temperature. After sedimentation at 3350 × g (3 min at 4 °C), the pellet was washed twice with ice-cold wash buffer (150 mm NaCl, 20 mm Tris-HCl (pH 7.4)) and two times with ice-cold PBS (8000 × g, 3 min at 4 °C). Thereafter, the beads were incubated with 500 μl of PBS, containing thrombin (3.25 NIH units/ml) for 60 min at room temperature. For the elimination of thrombin, the beads were sedimented (8000 × g, 1 min at 4 °C), and the supernatant was incubated with a benzamidine bead suspension for 10 min at 4 °C and sedimented at 8000 × g for 1 min at 4 °C. Afterward, an aliquot of the supernatant was subjected to a 12.5% SDS-PAGE, and the protein concentration was determined by densitometry by using Photoshop 7.0 software.Cell Culture and Cytotoxicity Assays—Vero cells were cultivated in DMEM (Invitrogen) containing 10% heat-inactivated fetal calf serum, 1 mm sodium pyruvate, 2 mm l-glutamate, and 0.1 mm nonessential amino acids. HeLa cells were cultivated in minimum Eagle's medium (Invitrogen) supplemented with 10% fetal calf serum and 2 mm l-glutamate. NIH 3T3 cells and J774.A1 macrophage-like cells were cultivated in DMEM (Invitrogen) containing 10% fetal calf serum, 4 mm l-glutamate, 1.5 g/liter sodium pyruvate, and 4.5 g/liter glucose. All cell lines were cultivated at 37 °C and 5% CO2. Cells were trypsinized and reseeded for at most 15–20 times. For cytotoxicity experiments, cells were seeded in culture dishes and incubated with the respective toxin in complete medium.Cell Vitality After Intoxication—Cell viability was determined by using the CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay from Promega, referred to as the MTS assay according to the tetrazolium compound (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS)). Subconfluently grown J774.A1 macrophage-like cells (96-well plate) were incubated at 37 °C for 7 h with the toxins or left untreated for control. Subsequently, 20 μl of the MTS reagent was added, and cells were further incubated for an additional 2 h. The absorption at 490 nm was measured using an enzyme-linked immunosorbent assay reader.Immunocytochemistry and Microinjection—Cells were seeded on coverslips and cultivated for at least 16 h. For actin staining, cells were washed with prewarmed PBS and fixed with 4% of paraformaldehyde for 20 min, washed twice with PBS, permeabilized with 0.2% (v/v) Triton X-100 in PBS, and washed again twice with PBS. Subsequently, cells were incubated with Alexa594-conjugated phalloidin (optional with 4,6-diamidino-2-phenylindole) for 30 min, washed with PBS, and embedded on glass slips with ProLong© Gold antifade reagent according to the manufacturer's manual. Cells were visualized by using a Zeiss Axiovert 200 (Oberkochen, Germany) with Cairn Optoscan monochromator system (Faversham, UK) and Photometrics CoolSNAPHQ CCD camera (Tucson, AZ). Images were processed by using Metamorph 6.1 software from Visitron (Munich, Germany). For microinjection, cells were seeded at a subconfluent density on glass coverslips (Cellocate; Eppendorf, Germany) and cultured overnight. Microinjection buffer (20 mm Tris-HCl (pH 7.5)), C2IN-C/SpvB(pro7) (50 nm in microinjection buffer), or C/SpvB (50 nm in microinjection buffer) was microinjected into Vero cells with an Eppendorf 5242 microinjector.ADP-ribosylation Assay—The ADP-ribosylation reaction was performed for 30 min at 37 °C in a buffer containing 50 mm Hepes buffer (pH 7.4), 2 mm MgCl2, 150 μm [adenylate-32P]NAD, 6 μm of β/γ-actin, and 1 μm of the enzymes C2I, C/SpvB, and C2IN-C/SpvB. The reaction was stopped by addition of SDS sample buffer and heating of the samples for 5 min at 95 °C. Radiolabeled proteins were detected by SDS-PAGE and analyzed by PhosphorImaging. For ADP-ribosylation of cell lysates, 10 μg of whole-cell lysate were used, and ADP-ribosylation was performed as described above with different amounts of toxins. The samples were run on SDS-PAGE, and [32P]ADP-ribosylated proteins were detected by autoradiography with a PhosphorImager from Amersham Biosciences. For actin-ADP-ribosylation of cell lysates, cultured cells were lysed with 20 mm Tris-HCl (pH 7.5), 1 mm EDTA, 1 mm dithiothreitol, 5 mm MgCl2, and Complete protease inhibitor (according to the manufacturer's manual). The complete cell protein samples (20–50 μg of protein) were incubated together with 10 μm biotinylated NAD and 300 ng of C2I at 30 °C for 30 min. The reaction was stopped by addition of 5× SDS sample buffer (625 mm Tris-HCl (pH 6.8), 20% SDS, 8.5% glycerol, 0.2% bromphenol blue, 100 mm dithiothreitol) and subsequent heating of the samples for 5 min at 95 °C. Biotin-ADP-ribosylated proteins were detected by immunoblot analysis with peroxidase-coupled streptavidin.SDS-PAGE and Immunoblot Analysis—SDS-PAGE was performed according to the method of Laemmli (23Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (206024) Google Scholar). The proteins were stained with Coomassie Brilliant Blue R-250. For immunoblot analysis, the proteins were transferred onto a nitrocellulose membrane. The membrane was blocked for 30 min with 5% nonfat dry milk in PBS containing 0.05% Tween 20 (PBS-T), and the proteins were probed with anti-C2IN antiserum (rabbit, 1:5000 in PBS-T). After washing with PBS-T, the blot was incubated for 1 h with an anti-rabbit antibody coupled to horseradish peroxidase (1:2000 in PBS-T). The membrane was washed again, and proteins were visualized using the ECL system according to the manufacturer's instructions.Assay for Toxin Binding to the Cell Surface—Confluently grown Vero cells were incubated in serum-free DMEM at 4 °C with the toxins (C2IIa + C2I; C2IIa + fusion toxins) to allow the binding of the proteins to the cell surface. Before adding to the cells, the toxin components were preincubated on ice for 15 min. As a control, the single components of the binary toxins (C2IIa, C2I, fusion toxins) were taken. Following a washing step with serum-free DMEM, cells were lysed in RIPA buffer, and equal amounts of protein (50 μg of each sample) were subjected to SDS-PAGE. The proteins were transferred from the gel onto a nitrocellulose membrane using the semi-dry system. The membranes were blocked overnight with 5% nonfat dry milk in PBS containing 0.05% Tween 20 (PBS-T) followed by a 1.5-h incubation with the antiserum (anti-C2IN, rabbit, 1:5000). After washing with PBS-T, the membrane was incubated with a donkey anti-rabbit antibody coupled to horseradish peroxidase (1:2.000 in PBS-T) for 1 h and washed, and the C2IN-containing proteins were detected using the ECL system.Assay for Translocation of Toxins across the Cell Membrane—Subconfluently grown HeLa and Vero cells were treated in serum-free DMEM for 30 min at 37 °C with 100 nm bafilomycin A1 to inhibit the physiological uptake pathway for C2 toxin. Subsequently, the cells were incubated for 20 min at 4 °C with the toxins (C2I + C2IIa; C2IN/C/SpvB + C2IIa; preincubated for 15 min at 4 °C to allow complex formation) and for 15 min at 4 °C in the presence of bafilomycin to allow the binding of the toxins to the cell surface. The medium was removed, and warm acidic medium (37 °C, pH 4.6, containing bafilomycin A1) was added to the cells for 5 min at 37 °C. This acidic pulse mimics the endosomal conditions. Finally, the acidic medium was replaced by neutral medium (pH 7.5) containing bafilomycin A1, and cells were incubated at 37 °C. The cytopathic effect of the toxin, i.e. cell rounding, was detected by taking pictures of the cells after various incubation periods.Reproducibility of the Experiments and Statistics—All experiments were performed independently at least three times. Results from representative experiments are shown in the figures. Values (n ≥ 3) are calculated as mean ± S.D. using the SigmaPlot software.RESULTSCloning, Expression, and Biochemical Characterization of the C2IN-C/SpvB Fusion Toxin—To deliver the catalytic domain of the ADP-ribosyltransferase SpvB (C/SpvB) into the cytosol of eukaryotic cells, we used a C2 toxin-based transport mechanism. To this end, a fusion toxin was cloned, consisting of amino acid residues 1–225 of the C2I enzyme component (C2IN, adaptor for the interaction with the C2IIa transport component) and the C/SpvB domain, encompassing residues 374–591 of the SpvB protein (Fig. 1A). The wild type S. enterica SpvB protein harbors a region of seven-proline residues between the N- and C-terminal domains, according to the amino acid residues 367–373 in the SpvB protein. We included this seven-proline region into the C2IN-C/SpvB fusion toxin between the two domains C2IN and C/SpvB (C2IN-C/SpvB(pro7) in the following referred to as C2IN-C/SpvB). To compare the fusion toxin with the C/SpvB domain and the C. botulinum C2I ADP-ribosyltransferase, the proteins C/SpvB, C2I, and C2IN-C/SpvB were expressed as recombinant glutathione S-transferase fusion proteins in E. coli cells and purified by affinity chromatography and subsequent cleavage of the GST domain with thrombin. The resulting proteins were analyzed by SDS-PAGE and by an immunoblot analysis with an antibody raised against the C2IN protein. Fig. 1B shows the Coomassie-stained proteins C/SpvB (∼25 kDa), C2I (∼50 kDa), and the C2IN-C/SpvB fusion protein (∼51 kDa). The specific antibody against C2IN recognized C2I and the C2IN-C/SpvB fusion toxin but not C/SpvB (Fig. 1C). In preparations of the C2IN-C/SpvB protein, a degradation product of ∼26 kDa (Fig. 1, B and C) was also recognized by the anti-C2I antiserum, i.e. this protein was C2IN or contained a part of C2IN. Matrix-assisted laser desorption ionization time-of-flight analysis of the two degradation products confirmed that one protein contained the C2IN peptide and the other contained the C/SpvB peptide.Next, we tested whether the C2IN-C/SpvB fusion toxin was enzymatically active. We performed an ADP-ribosylation assay with non-muscle (β/γ) actin as a substrate, [32P]NAD as a co-substrate, and the mono-ADP-ribosyltransferases C/SpvB, C2I, and C2IN-C/SpvB as enzymes. The autoradiography of radiolabeled, i.e. ADP-ribosylated actin, is shown in Fig. 1D. This result demonstrates that the C2IN-C/SpvB fusion toxin was active in vitro. To test the activity of C2IN-C/SpvB in intact cells, the protein was microinjected into Vero cells. Directly and after the indicated incubation periods, pictures from identical cells were taken (Fig. 1E). Injection of control buffer as a control for the microinjection procedure did not influence the morphology of cells (Fig. 1E, left panel). When the C/SpvB protein as a positive control was injected, cells rounded up and finally detached from the coverslip (Fig. 1E, middle panel). Similarly, cells microinjected with C2IN-C/SpvB rounded up within a period of 60 min (Fig. 1E, right panel), indicating that the C2IN-C/SpvB fusion toxin destroyed the structure of the actin cytoskeleton in infected cells.Cellular Uptake Mechanism of the C2IN-C/SpvB Fusion Toxin—Next, we tested whether the fusion toxin C2IN-C/SpvB was delivered into cells by the transport component C2IIa. To this end, subconfluently growing Vero cells were incubated with the C2IN-C/SpvB fusion toxin (1 μg/ml) together with C2IIa (2 μg/ml). As a positive control, the C. botulinum C2 toxin (C2I + C2IIa) was used. As further controls, cells were incubated with the single components C2I (200 ng/ml), C2IN-C/SpvB (1 μg/ml), and C2IIa (1 μg/ml) and without any protein. Cells were incubated for 3 h at 37°C; pictures were taken, and the morphology of the cells was determined as an end point for the cytopathic activity of the individual protein combinations (Fig. 2A). Only such cells, which have been treated with the combination of C2IIa plus C2IN-C/SpvB or C2IIa plus C2I, showed a round morphology. As expected, treatment of the cells with the single components C2IIa, C2I, or C2IN-C/SpvB did not induce any morphological alterations, indicating that for transport of the C2IN-C/SpvB fusion toxin into cells C2IIa is essentially needed. After a 3-h incubation period, the majority of the cells were rounded up (Fig. 2A). This change in morphology was comparable with the rounding of cells, which have been treated with C2I and C2IIa.FIGURE 2Cytopathic effect of the C2IN-C/SpvB fusion toxin on Vero cells. A, morphologic effects of the C2IN-C/SpvB fusion toxin. Vero cells were incu" @default.
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• W2014404744 date "2007-04-01" @default.
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• W2014404744 title "A Cell-permeable Fusion Toxin as a Tool to Study the Consequences of Actin-ADP-ribosylation Caused by the Salmonella enterica Virulence Factor SpvB in Intact Cells" @default.
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