Dominant-negative Inhibitors of the Clostridium perfringens ϵ-Toxin
2009; Elsevier BV; Volume: 284; Issue: 43 Linguagem: Inglês
10.1074/jbc.m109.021782
ISSN1083-351X
AutoresTeal M. Pelish, Mark S. McClain,
Tópico(s)Nanopore and Nanochannel Transport Studies
ResumoThe Clostridium perfringens ϵ-toxin is responsible for a severe, often lethal intoxication. In this study, we characterized dominant-negative inhibitors of the ϵ-toxin. Site-specific mutations were introduced into the gene encoding ϵ-toxin, and recombinant proteins were expressed in Escherichia coli. Paired cysteine substitutions were introduced at locations predicted to form a disulfide bond. One cysteine in each mutant was introduced into the membrane insertion domain of the toxin; the second cysteine was introduced into the protein backbone. Mutant proteins with cysteine substitutions at amino acid positions I51/A114 and at V56/F118 lacked detectable cytotoxic activity in a MDCK cell assay. Cytotoxic activity could be reconstituted in both mutant proteins by incubation with dithiothreitol, indicating that the lack of cytotoxic activity was attributable to the formation of a disulfide bond. Fluorescent labeling of the cysteines also indicated that the introduced cysteines participated in a disulfide bond. When equimolar mixtures of wild-type ϵ-toxin and mutant proteins were added to MDCK cells, the I51C/A114C and V56C/F118C mutant proteins each inhibited the activity of wild-type ϵ-toxin. Further analysis of the inhibitory activity of the I51C/A114C and V56C/F118C mutant proteins indicated that these proteins inhibit the ability of the active toxin to form stable oligomeric complexes in the context of MDCK cells. These results provide further insight into the properties of dominant-negative inhibitors of oligomeric pore-forming toxins and provide the basis for developing new therapeutics for treating intoxication by ϵ-toxin. The Clostridium perfringens ϵ-toxin is responsible for a severe, often lethal intoxication. In this study, we characterized dominant-negative inhibitors of the ϵ-toxin. Site-specific mutations were introduced into the gene encoding ϵ-toxin, and recombinant proteins were expressed in Escherichia coli. Paired cysteine substitutions were introduced at locations predicted to form a disulfide bond. One cysteine in each mutant was introduced into the membrane insertion domain of the toxin; the second cysteine was introduced into the protein backbone. Mutant proteins with cysteine substitutions at amino acid positions I51/A114 and at V56/F118 lacked detectable cytotoxic activity in a MDCK cell assay. Cytotoxic activity could be reconstituted in both mutant proteins by incubation with dithiothreitol, indicating that the lack of cytotoxic activity was attributable to the formation of a disulfide bond. Fluorescent labeling of the cysteines also indicated that the introduced cysteines participated in a disulfide bond. When equimolar mixtures of wild-type ϵ-toxin and mutant proteins were added to MDCK cells, the I51C/A114C and V56C/F118C mutant proteins each inhibited the activity of wild-type ϵ-toxin. Further analysis of the inhibitory activity of the I51C/A114C and V56C/F118C mutant proteins indicated that these proteins inhibit the ability of the active toxin to form stable oligomeric complexes in the context of MDCK cells. These results provide further insight into the properties of dominant-negative inhibitors of oligomeric pore-forming toxins and provide the basis for developing new therapeutics for treating intoxication by ϵ-toxin. The Clostridium perfringens ϵ-toxin is one of the most potent bacterial toxins (1Gill D.M. Microbiol. Rev. 1982; 46: 86-94Crossref PubMed Google Scholar, 2Minami J. Katayama S. Matsushita O. Matsushita C. Okabe A. Microbiol. Immunol. 1997; 41: 527-535Crossref PubMed Scopus (126) Google Scholar). The ϵ-toxin can lead to a fatal enterotoxemia characterized by widespread vascular permeability and edema in the heart, lungs, brain, and kidneys (3Gardner D.E. N. Z. Vet. J. 1972; 20: 167-168Crossref PubMed Scopus (1) Google Scholar, 4Adamson R.H. Ly J.C. Fernandez-Miyakawa M. Ochi S. Sakurai J. Uzal F. Curry F.E. Infect. Immun. 2005; 73: 4879-4887Crossref PubMed Scopus (28) Google Scholar, 5Soler-Jover A. Dorca J. Popoff M.R. Gibert M. Saura J. Tusell J.M. Serratosa J. Blasi J. Martin-Satué M. Toxicon. 2007; 50: 530-540Crossref PubMed Scopus (48) Google Scholar, 6Worthington R.W. Mülders M.S. Onderstepoort J. Vet. Res. 1975; 42: 25-27PubMed Google Scholar). The disease most frequently affects livestock animals, though the toxin may also affect humans (7Gleeson-White M.H. Bullen J.J. Lancet. 1955; 268: 384-385Abstract PubMed Scopus (19) Google Scholar, 8Kohn J. Warrack G.H. Lancet. 1955; 268: 385Abstract PubMed Scopus (17) Google Scholar, 9Smith L.D.S. Williams B.L. The Pathogenic Anaerobic Bacteria. Third Ed. Charles C. Thomas, Springfield, Illinois1984Google Scholar). Because of its extreme potency and the possibility of intoxicating humans, the C. perfringens ϵ-toxin is considered a select agent by the United States Department of Health and Human Services. A vaccine currently is approved for veterinary use, though multiple immunizations are required to provide long-term immunity (10Cameron C.M. Onderstepoort J. Vet. Res. 1980; 47: 287-289PubMed Google Scholar, 11de la Rosa C. Hogue D.E. Thonney M.L. J. Anim. Sci. 1997; 75: 2328-2334Crossref PubMed Scopus (36) Google Scholar, 12Uzal F.A. Bodero D.A. Kelly W.R. Nielsen K. Vet. Rec. 1998; 143: 472-474Crossref PubMed Scopus (35) Google Scholar, 13Uzal F.A. Kelly W.R. Vet. Rec. 1998; 142: 722-725Crossref PubMed Scopus (27) Google Scholar). There also is an antitoxin approved for veterinary use. However, in the event that an animal exhibits symptoms of intoxication by ϵ-toxin, it is typically too late for the current antitoxin to be effective, and use of the antitoxin is typically limited to prophylactic treatment of unvaccinated animals within a herd (14Beers M. Berkow R. The Merck Manual of Diagnosis and Therapy. 17th Edition. Merck and Co., Inc., 2005Google Scholar). There is no treatment currently approved for use in humans. Thus, alternative countermeasures are needed that inhibit the activity of the toxin. One alternative method of countering the cytotoxic activity of bacterial toxins is through dominant-negative inhibitors. Dominant-negative inhibitors are non-cytotoxic mutant forms of active toxins that are able to inhibit the activity of wild-type toxin when the two proteins are mixed together. Such dominant-negative inhibitors have been described for a diverse set of toxins, including Helicobacter pylori VacA (15Genisset C. Galeotti C.L. Lupetti P. Mercati D. 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In the case of VacA and protective antigen, the most extensively studied examples of toxins inhibited by dominant-negative mutants, the number of mutations that inactivate the toxins is substantially greater than the number of mutations that lead to a dominant-negative phenotype (16McClain M.S. Cao P. Iwamoto H. Vinion-Dubiel A.D. Szabo G. Shao Z. Cover T.L. J. Bacteriol. 2001; 183: 6499-6508Crossref PubMed Scopus (95) Google Scholar, 17Vinion-Dubiel A.D. McClain M.S. Czajkowsky D.M. Iwamoto H. Ye D. Cao P. Schraw W. Szabo G. Blanke S.R. Shao Z. Cover T.L. J. Biol. Chem. 1999; 274: 37736-37742Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 24Mourez M. Yan M. Lacy D.B. Dillon L. Bentsen L. Marpoe A. Maurin C. Hotze E. Wigelsworth D. Pimental R.A. Ballard J.D. Collier R.J. Tweten R.K. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 13803-13808Crossref PubMed Scopus (71) Google Scholar, 31McClain M.S. Cover T.L. Infect. 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Thus, a deeper understanding of the nature of the dominant-negative phenotype is needed. In this study, we sought to generate dominant-negative mutants of the ϵ-toxin. We hypothesized that mutations within the membrane insertion domain of ϵ-toxin, particularly mutations that are expected to restrict movement of this domain, would lead to dominant-negative inhibitors. We expressed wild-type and site-specific mutants of the ϵ-toxin as recombinant proteins in E. coli. The recombinant proteins were purified, and cytotoxicity was assessed using an established cell culture assay. Using this approach, we identified mutant proteins that inhibited the activity of wild-type ϵ-toxin in vitro and determined the mechanism of inhibition. The gene encoding ϵ-prototoxin, etxB, from C. perfringens type B strain ATCC3626 was PCR-amplified and cloned into plasmid pET22b (Novagen). This placed the etxB gene under the regulation of the bacteriophage T7 RNA polymerase and fused the N-terminal end of the prototoxin to the pelB leader peptide and the C-terminal end of the prototoxin to a His6 affinity tag (to aid in purification of the protein). A derivative plasmid that expressed a GFP-ϵ-toxin fusion protein was also constructed (5Soler-Jover A. Dorca J. Popoff M.R. Gibert M. Saura J. Tusell J.M. Serratosa J. Blasi J. Martin-Satué M. Toxicon. 2007; 50: 530-540Crossref PubMed Scopus (48) Google Scholar, 33Soler-Jover A. Blasi J. Gómez de Aranda I. Navarro P. Gibert M. Popoff M.R. Martin-Satué M. J. Histochem. Cytochem. 2004; 52: 931-942Crossref PubMed Scopus (63) Google Scholar, 34Dorca-Arevalo J. Soler-Jover A. Gibert M. Popoff M.R. Martin-Satue M. Blasi J. Vet. Microbiol. 2008; 131: 14-25Crossref PubMed Scopus (70) Google Scholar). The ϵ-prototoxin-expressing plasmid was transformed into an E. coli K12 expression strain, NovaBlue (DE3) (Novagen), along with the plasmid pLysE (encoding bacteriophage T7 lysozyme) and transformants were grown in broth supplemented with antibiotics to an optical density at 600 nm of 0.7. Isopropyl β-d-thiogalactopyranoside (IPTG) then was added to a final concentration of 1 mm to induce expression of the cloned gene, and the cultures were grown for another 3 h. The cells were collected by centrifugation, resuspended in 1:20th culture volume of B-PER Bacterial Protein Extraction Reagent (Pierce) supplemented with Complete Mini protease inhibitor mixture (EDTA-free, Roche Applied Sciences) and mixed for 10 min at room temperature. Omnicleave nuclease (Epicenter) was added to reduce the viscosity of the samples. The cell debris was pelleted, and the supernatant was recovered. The B-PER extracted material was diluted 4-fold with water and applied to a Q-Sepharose column. The ϵ-prototoxin-containing flow-through material was collected and applied to a Ni-nitrilotriacetic acid (NTA) affinity column (Qiagen). The Ni-NTA column was washed with a buffer composed of 20 mm sodium phosphate, 300 mm sodium chloride, and 20 mm imidazole (pH 8.0), and the ϵ-prototoxin was eluted in a buffer composed of 20 mm sodium phosphate, 300 mm sodium chloride, and 250 mm imidazole (pH 8.0). The identification of the ϵ-prototoxin in the purified sample was confirmed by immunoblotting with ϵ-toxin-specific monoclonal antibody 5B7 (35McClain M.S. Cover T.L. Infect. Immun. 2007; 75: 1785-1793Crossref PubMed Scopus (32) Google Scholar, 36Hauer P.J. Clough N.E. Dev. Biol. Stand. 1999; 101: 85-94PubMed Google Scholar). Protein concentrations were determined using micro-BCA (Pierce). The ϵ-prototoxin can be cleaved with trypsin to remove short peptides from both the N- and C-terminal ends of the protein to yield the active ϵ-toxin (2Minami J. Katayama S. Matsushita O. Matsushita C. Okabe A. Microbiol. Immunol. 1997; 41: 527-535Crossref PubMed Scopus (126) Google Scholar, 28Miyata S. Matsushita O. Minami J. Katayama S. Shimamoto S. Okabe A. J. Biol. Chem. 2001; 276: 13778-13783Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 35McClain M.S. Cover T.L. Infect. Immun. 2007; 75: 1785-1793Crossref PubMed Scopus (32) Google Scholar). Trypsin-coated agarose beads (Pierce) were washed and resuspended in 5 mm Tris, pH 7.5. Preparations containing the ϵ-prototoxin were incubated with trypsin-agarose at 37 °C for 60 min, the trypsin-coated beads were removed by centrifugation, and residual trypsin was inhibited by Complete Mini protease inhibitor mixture (Roche Applied Sciences). Conversion of the ϵ-prototoxin to ϵ-toxin was assessed based on SDS-PAGE and immunoblotting with anti-ϵ-toxin antibodies (36Hauer P.J. Clough N.E. Dev. Biol. Stand. 1999; 101: 85-94PubMed Google Scholar). Following precedent in the literature, the N-terminal amino acid of the mature ϵ-toxin (i.e. following trypsin treatment) is numbered 1 (35McClain M.S. Cover T.L. Infect. Immun. 2007; 75: 1785-1793Crossref PubMed Scopus (32) Google Scholar, 37Oyston P.C. Payne D.W. Havard H.L. Williamson E.D. Titball R.W. Microbiology. 1998; 144: 333-341Crossref PubMed Scopus (56) Google Scholar). MDCK cells in Leibovitz l-15 medium supplemented with 10% fetal bovine serum were added to multiwell plates (5 × 103 cells per well in 384-well dishes). ϵ-Toxin was added, and the cells then were incubated at 37 °C for 16 h. Cytotoxicity was determined by treating cells with resazurin (CellTiter Blue, Promega) at 37 °C for 4 h. To determine the effect of a reducing agent on the cytotoxic activity of wild-type ϵ-toxin and mutant proteins, toxin preparations were incubated at 45 °C for 1 h in the presence of 3 mm dithiothreitol (DTT). 2The abbreviations used are: DTTdithiothreitolTCEPTris(2-carboxyethyl)phosphineGFPgreen fluorescent proteinANOVAanalysis of varianceGAPDHglyceraldehyde-3-phosphate dehydrogenase. Samples then were diluted 12-fold upon addition to the medium overlying MDCK cell monolayers. The final toxin concentration was 6.5 nm, and the final DTT concentration was 0.25 mm. Cytotoxicity was determined as described above. dithiothreitol Tris(2-carboxyethyl)phosphine green fluorescent protein analysis of variance glyceraldehyde-3-phosphate dehydrogenase. Mutations were introduced into the cloned etxB gene using the QuikChange multi site-directed mutagenesis kit (Stratagene). To determine whether cysteine residues introduced into the ϵ-toxin formed disulfide bonds, proteins were reacted with iodoacetamide, to block free sulfhydryls, followed by reduction and labeling of sulfhydryls with a fluorescent marker (38Minard K.I. Carroll C.A. Weintraub S.T. Mc-Alister-Henn L. Free Radic Biol. Med. 2007; 42: 106-117Crossref PubMed Scopus (11) Google Scholar, 39Yano H. Anal. Chem. 2003; 75: 4682-4685Crossref PubMed Scopus (16) Google Scholar). Purified ϵ-toxin preparations (20 μg) were incubated with iodoacetamide (2.7 mm final concentration, to block cysteines not participating in disulfide bonds) and Alexa Fluor 680 succinimidyl ester (0.5 mm final concentration, to fluorescently label the proteins) in the presence of 1% SDS and 4 m urea. The reactions were incubated at room temperature in the dark for 1 h. The labeled proteins were separated from unincorporated dye and iodoacetamide using a Zeba spin column (Pierce). Each protein sample then was divided into two aliquots. To the first aliquot, TCEP was added (30 mm final concentration) to reduce disulfide bonds; water was added to the second aliquot as a nonreduced control. Reactions were incubated at 55 °C for 30 min. Proteins were separated from reaction components using a Zeba spin column, with the protein sample collected into a tube containing Dye Light 800 maleimide to fluorescently label free sulfhydryls (40 μm final concentration). Reactions were incubated at room temperature for 1 h. Proteins were separated from reaction components using a Zeba spin column, separated by SDS-PAGE, and analyzed using a LiCor Odyssey fluorescent scanner. Purified proteins (Sigma) labeled in control reactions included equine myoglobin (no disulfide bonds, Ref. 40Evans S.V. Brayer G.D. J. Mol. Biol. 1990; 213: 885-897Crossref PubMed Scopus (266) Google Scholar), E. coli thioredoxin (1 disulfide bond, Ref. 41Katti S.K. LeMaster D.M. Eklund H. J. Mol. Biol. 1990; 212: 167-184Crossref PubMed Scopus (535) Google Scholar), soybean trypsin inhibitor (2 disulfide bonds, Ref. 42Koide T. Ikenaka T. Eur. J. Biochem. 1973; 32: 417-431Crossref PubMed Scopus (170) Google Scholar), and hen egg lysozyme (4 disulfide bonds, Ref. 43Blake C.C. Koenig D.F. Mair G.A. North A.C. Phillips D.C. Sarma V.R. Nature. 1965; 206: 757-761Crossref PubMed Scopus (1260) Google Scholar). The fluorescent intensities at 800 nm (FI800) and 680 nm (FI680) were determined for each protein using LiCor Odyssey software. Results are expressed as a fluorescence ratio (FR) according to Equation 1. FR=(FI800)+TCEP/(FI680)+TCEP(FI800)-TCEP/(FI680)-TCEP(Eq. 1) Plasmid DNA capable of expressing Etx-I51C/A114C or Etx-V56C/F118C was transformed into E. coli K12 strain HMS174 (DE3) pLysS (Novagen). Transformants were cultured according to the manufacturer's instructions in methionine-restricted, chemically defined medium (Overnight Express Auto-Induction System 2, Novagen) supplemented with 2 mm l-photomethionine (Pierce). The Etx-I51C/A114C and Etx-V56C/F118C mutant proteins containing l-photomethionine were purified as described above. Protein samples in SDS sample buffer were heated in a boiling water bath for 5 min before analysis by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were developed using anti-GFP (Santa Cruz Biotechnology, SC-9996) or anti-GAPDH (Abcam, ab9484) antibodies followed by horseradish peroxidase-conjugated goat anti-mouse antibody. SuperSignal West Femto or SuperSignal West Pico substrates (Pierce) were used for enhanced chemiluminescent detection. Statistical analyses were performed using SigmaStat software. The DNA sequences of the genes encoding wild-type and mutant epsilon toxins were determined using the Vanderbilt University DNA Sequencing shared resource. Plasmid DNA capable of expressing the ϵ-prototoxin (or ϵ-toxin) is considered a select agent by the U.S. Department of Health and Human Services.
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