Najime GORD-NOSHAHRI, Mohsen Fathı NAJAFI, Ali MAKHDOUMI, Behjat MAJIDI, Mohsen MEHRVARZ

Identification and cloning of highly epitopic regions of Clostridium novyi alpha toxin

The aim of the present study is to provide a strategy for predicting the production of antigenic regions of Clostridium novyi alpha toxin. The selection is based on B-cell epitopes and MHCII binding protein by immunoinformatics tools. The study resulted in identifying antigenic regions in the beginning and middle (3-17 and 965-997 amino acid residues) as suitable binders to MHCII and the carboxyl terminal of the protein (1800-1958 amino acid residues) in B-cell epitopes. The appropriate region in B-cell epitopes was chosen for cloning. The presence of recombinant protein was detected with immunological methods. Subsequently, BALB/C mice were immunized with the recombinant protein and alpha toxin, and the antibodies produced were evaluated by dot-blot and ELISA tests. After cloning the highly antigenic region (1799-1966 amino acid residues), the results of immunological tests showed that the recombinant protein reacts with antitoxin antibodies. Antirecombinant protein has a higher affinity to the alpha toxin than antialpha toxin. Thus, the carboxyl terminal of the protein (1799-1966 amino acid residues) in B-cell epitopes could be a proper candidate for a peptide vaccine against alpha toxin.

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Atassi MZ, Dolimbek BZ, Jankovic J, Steward LE, Aoki KR (2011). Regions of botulinum neurotoxin A light chain recognized by human anti-toxin antibodies from cervical dystonia patients immunoresistant to toxin treatment. The antigenic structure of the active toxin recognized by human antibodies. Immunobiology 216: 782-792.
Atassi MZ, Dolimbek BZ, Steward LE, Aoki KR (2012). Antibodies against a synthetic peptide designed to mimic a surface area of the H chain of botulinum neurotoxin A. Immunol Lett 142: 20-27.
Barlow D, Edwards M, Thornton J (1986). Continuous and discontinuous protein antigenic determinants. Nature 322: 747-748.
Bette P, Oksche A, Mauler F, Eichel-Streiber C, Popoff M, Habermann E (1991). A comparative biochemical, pharmacological and immunological study of Clostridium novyi α-toxin, C. difficiletoxin B and C. sordellii lethal toxin. Toxicon 29: 877-887.
Busch C, Schömig K, Hofmann F, Aktories K (2000). Characterization of the catalytic domain of Clostridium novyi alpha-toxin. Infect Immun 68: 6378-6383.
Cheng HR, Jiang N (2006). Extremely rapid extraction of DNA from bacteria and yeasts. Biotechnol Lett 28: 55-59.
de Paz LEC, Resin A, Howard KA, Sutherland DS, Wejse PL (2011). Antimicrobial effect of chitosan nanoparticles on Streptococcus mutans biofilms. Appl Environ Microb 77: 3892-3895.
Fox JL, Klass M (1989). Antigens produced by recombinant DNA technology. Clin Chem 35: 1838-1842.
Gasteiger E, Hoogland C, Gattiker A, Wilkins MR, Appel RD, Bairoch A (2005). Protein identification and analysis tools on the ExPASy server. In: Walker JM, editor. The Proteomics Protocols Handbook. Totowa, NJ, USA: Humana Press, pp. 571-607.
Guan Z, Johnston NC, Aygun-Sunar S, Daldal F, Raetz CR, Goldfine H (2011). Structural characterization of the polar lipids of Clostridium novyi NT. Further evidence for a novel anaerobic biosynthetic pathway to plasmalogens.BBA-Mol Cell Biol L 1811: 186-193.
Haesebrouck F, Pasmans F, Chiers K, Maes D, Ducatelle R, Decostere A (2004). Efficacy of vaccines against bacterial diseases in swine: what can we expect? Vet Microbiol 100: 255-268.
Hatheway CL (1990). Toxigenic clostridia. Clin Microbiol Rev 3: 66.
Hofmann F, Herrmann A, Habermann E, von Eichel-Streiber C (1995). Sequencing and analysis of the gene encoding the α-toxin of Clostridium novyi proves its homology to toxins A and B of Clostridium difficile. Mol Gen Genet 247: 670-679.
Ingale A (2010). Antigenic epitopes prediction and MHC binder of a paralytic insecticidal toxin (ITX-1) of Tegenaria agrestis (hobo spider). Bioinformatics 2: 97-103.
Irving MB, Pan O, Scott JK (2001). Random-peptide libraries and antigen-fragment libraries for epitope mapping and the development of vaccines and diagnostics. Curr Opin Chem Biol 5: 314-324.
Kolaskar A, Tongaonkar PC (1990). A semi-empirical method for prediction of antigenic determinants on protein antigens. FEBS Lett 276: 172-174.
Kringelum JV, Lundegaard C, Lund O, Nielsen M (2012). Reliable B cell epitope predictions: impacts of method development and improved benchmarking. PLoS Comput Biol 8: e1002829.
Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.
Lovell SC, Davis IW, Arendall WB, de Bakker PI, Word JM, Prisant MG, Richardson JS, Richardson DC (2003). Structure validation by Cα geometry: φ, ψ and Cβ deviation. Proteins 50: 437-450.
McGregor D, Molloy P, Cunningham C, Harris W (1994). Spontaneous assembly of bivalent single chain antibody fragments in Escherichia coli. Mol Immunol 31: 219-226.
McGuigan CC, Penrice GM, Gruer L, Ahmed S, Goldberg D, Black M, Salmon JE, Hood J (2002). Lethal outbreak of infection with Clostridium novyi type A and other spore-forming organisms in Scottish injecting drug users. J Med Microbiol 51: 971-977.
Modrow S, Hahn BH, Shaw GM, Gallo RC, Wong-Staal F, Wolf H (1987). Computer-assisted analysis of envelope protein sequences of seven human immunodeficiency virus isolates: prediction of antigenic epitopes in conserved and variable regions. J Virol 61: 570-578.
Nagahama M, Kobayashi K, Oda M (2012). Glycosylating Toxin of Clostridium Perfringens. Rijeka, Croatia: InTech.
Nijland R, Lindner C, Van Hartskamp M, Hamoen LW, Kuipers OP (2007). Heterologous production and secretion of Clostridium perfringens β-toxoid in closely related Gram-positive hosts. J Biotechnol 127: 361-372.
Oksche A, Nakov R, Habermann E (1992). Morphological and biochemical study of cytoskeletal changes in cultured cells after extracellular application of Clostridium novyi alpha-toxin. Infect Immun 60: 3002-3006.
Roy A, Kucukural A, Zhang Y (2010). I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 5:725-738.
Ru Z (2012). Creating and use of a new experimental preclinical HLA transgenic mice model to mapping HLA-restricted T cells epitopes for polyepitopes vaccine design. PhD, Université Paris Sud, Paris, France.
Saha S, Raghava G (2004). BcePred: prediction of continuous B-cell epitopes in antigenic sequences using physico-chemical properties. In: Nicosia G, Cutello V, Bentley PJ, Timmis J, editors. Artificial Immune Systems. Berlin, Germany: Springer-Verlag, pp. 197-204.
Sambrook J, Russell DW, Russell DW (2001). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY, USA: Cold Spring Harbor Laboratory Press.
Sarma P, Purkayastha S, Madan T, Sarma PU (2000). Expression of an epitopic region of AspfI, an allergen/antigen/cytotoxin of Aspergillus fumigatus. Immunol Lett 70: 151-155.
Tomar N, De RK (2014). Immunoinformatics: a brief review. Methods Mol Biol 1184: 23-55.
Towbin H, Gordon J (1984). Immunoblotting and dot immunobinding- current status and outlook. J Immunol Methods 72: 313-340
von Eichel-Streiber C, Boquet P, Sauerborn M, Thelestam M (1996). Large clostridial cytotoxins- a family of glycosyltransferases modifying small GTP-binding proteins. Trends Microbiol 4: 375-382.
Wang P, Sidney J, Dow C, Mothe B, Sette A, Peters B (2008). A systematic assessment of MHC class II peptide binding predictions and evaluation of a consensus approach. PLoS Comput Biol 4: e1000048.