In Silico Analysis of a Multi-subunit Immunogen, Targeting Virulence Factors of Enterohemorrhagic Escherichia coli

Document Type : Original Article


Department of Biological Sciences,, Faculty of Sciences, Imam Hossein University, Tehran, Iran


Enterohemorrhagic Escherichia coli (EHEC) strains are foodborne pathogens with importance in public health. The lack of effective clinical treatment, sequelae after infection and mortality rate in humans confirms the essential need for prophylactic and vaccines approaches. EspA, Tir, HcpA and Stx2 are major virulence factors for adherence and toxicity of EHEC, so an appropriate tetravalent immunogen consist of toxin subunit and crucial colonization factors was selected and constructed. Bioinfo[1]rmatic analyses of recombinant construction such as sequences choosing and optimizing, mRNA folding, physicochemical property in 2D and 3D structures, besides other immunoinformatics data like B-cell and T-cell epitopes and allergenicity of chimera were some reported according to the reliable servers. In silico assessment of the chimeric proteins demonstrated the desired model has a proper mRNA features, besides acceptable stability and solubility. This model is close to native proteins topologically, and all domains were found to have a high antigenic competency and surface accessibility. These results can be beneficial for the development of a chimeric immunogen against adherence and toxicity of EHEC in an animal model application.


  1. Nazarian, S., Gargari, S.L.M., Rasooli, I., Amani, J., Bagheri, S., Alerasool, M., An in silico chimeric multi subunit vaccine targeting virulence factors of enterotoxigenic Escherichia coli (ETEC) with its bacterial inbuilt adjuvant. J Microbiol Methods, 2012, Vol. 90, pp. 36-45.
  2. Babiuk, S., Asper, D.J., Rogan, D., Mutwiri, G.K., Potter, A.A., Subcutaneous and intranasal immunization with type III secreted proteins can prevent colonization and shedding of Escherichia coli O157: H7 in mice. Microb Pathog, 2008, Vol. 45, pp. 7-11.
  3. Larrie-Bagha, S.M., Rasooli, I., Mousavi-Gargari, S.L., Rasooli, Z., Nazarian, S., Passive immunization by recombinant ferric enterobactin protein (FepA) from Escherichia coli O157. Iran J Microbiol, 2013, Vol. 5, p. 113.
  4. Ishikawa, S., Kawahara, K., Kagami, Y., Isshiki, Y., Kaneko, A., Matsui, H., Okada, N., Danbara, H., Protection against Shiga toxin 1 challenge by immunization of mice with purified mutant Shiga toxin 1. Infect Immun, 2003, Vol. 71, pp. 3235-3239.
  5. Konadu, E., Donohue-Rolfe, A., Calderwood, S.B., Pozsgay, V., Shiloach, J., Robbins, J.B., Szu, S.C., Syntheses and Immunologic Properties of Escherichia coli O157 O-Specific Polysaccharide and Shiga Toxin 1 B Subunit Conjugates in Mice. Infect Immun, 1999, Vol. 67, pp. 6191–6193.
  6. Rad, H.S., Mousavi, S.L., Rasooli, I., Amani, J.,, Nadooshan, M.R.J., EspA-Intimin chimeric protein, a candidate vaccine against Escherichia coli O157: H7. Iran J Microbiol, 2013, Vol. 5, pp. 620–627.
  7. Amani, J., Mousavi, S.L., Rafati, S., Salmanian, A.H., Immunogenicity of a plant-derived edible chimeric EspA, Intimin and Tir of Escherichia coli O157: H7 in mice. Plant Sci, 2011, Vol. 180, pp. 620-627.
  8. Gu, J., Liu, Y., Yu, S., Wang, H., Wang, Q., Yi, Y., Zhu, F., Yu, X.J., Zou, Q., Mao, X., Enterohemorrhagic Escherichia coli trivalent recombinant vaccine containing EspA, intimin and Stx2 induces strong humoral immune response and confers protection. Microbes Infect, 2009, Vol. 11, pp. 835-841.
  9. Frankel, G., Phillips, A.D., Rosenshine, I., Dougan, G., Kaper, J.B., Knutton, S., Enteropathogenic and enterohaemorrhagic Escherichia coli: more subversive elements. Mol Microbiol, 1998, Vol. 30, pp. 911–921.
  10. Xicohtencatl-Cortes, J., Monteiro-Neto, V., Ledesma, M.A., Jordan, D.M., Francetic, O., Kaper, J.B., Puente, J.L., Girón, J.A., Intestinal adherence associated with type IV pili of enterohemorrhagic Escherichia coli O157: H7. J Clin Invest, 2007, Vol. 117, pp. 3519–3529.
  11. Doytchinova, I.A., Flower, D.R., VaxiJen: a server for prediction of protective antigens, tumour antigens and subunit vaccines. BMC Bioinformatics, 2007, Vol. 8, pp. 4-10.
  12. Zuker, M., Stiegler, P., Optimal computer folding of large RNA sequences using thermodynamics and auxiliary information. Nucleic Acids Res, 1981, Vol. 91, pp. 33–48.
  13. Reuter, J.S., Mathews, D.H., RNAstructure: software for RNA secondary structure prediction and analysis. BMC bioinformatics, 2010, Vol. 11, pp. 129-135.
  14. Gasteiger, E., Hoogland, C., Gattiker, A., Duvaud, S.E., Wilkins, M.R., Appel, R.D., Bairoch, A., Protein identification and analysis tools on the ExPASy server. Methods Mol Biol, 2005, pp. 571-607.
  15. Smialowski, P., Doose, G., Torkler, P., Kaufmann, S., Frishman, D., PROSO II–a new method for protein solubility prediction. FEBS J, 2012, Vol. 279, pp. 2192-2200.
  16. Rost, B., Sander, C., Schneider, R., PHD-an automatic mail server for protein secondary structure prediction. Bioinformatics, 1994, Vol. 10, pp. 53–60.
  17. Sen, T.Z., Jernigan, R.L., Garnier, J., Kloczkowski, A. GOR V server for protein secondary structure prediction. Bioinformatics, 2005, Vol. 21, pp. 2787-2788.
  18. Cheng, J., Randall, A.Z., Sweredoski, M.J., Baldi, P., SCRATCH: a protein structure and structural feature prediction server. Nucleic Acids Res, 2005, Vol. 33, pp. 72-76.
  19. McGuffin, L.J., Bryson, K., Jones, D.T., The PSIPRED protein structure prediction server. Bioinformatics, 2000, Vol. 16, pp. 404–405.
  20. Schwede, T., Kopp, J., Guex, N., Peitsch, M.C., SWISS-MODEL: An automated protein homology-modeling server. Nucleic Acids Res, 2003, Vol. 31, pp. 3381–3385.
  21. Roy, A., Kucukural, A., Zhang, Y., I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc, 2010, Vol. 5, pp. 725–738.
  22. Goodsell, D.S., Representing structural information with RasMol. Curr Protoc Bioinformatics, 2005, pp.4-5.
  23. Wiederstein, M., Sippl, M.J., ProSA-web: interactive web service for the recognition of errors in three-dimensional structures of proteins. Nucleic Acids Res, 2007, Vol. 35, pp. 407-410.
  24. MacArthur, M.W., Laskowski, R.A., Thornton, J.M., Knowledge-based validation of protein structure coordinates derived by X-ray crystallography and NMR spectroscopy. Curr Opin Struct Biol, 1994, Vol. 4, pp. 731-737.
  25. Lovell, S.C., Davis, I.W., Arendall, W.B., de Bakker, P.I., Word, J.M., Prisant, M.G., Richardson, J.S., Richardson, D.C., Structure validation by Cα geometry:ϕ, ψ and Cβ deviation. Proteins, 2003, Vol. 50, pp. 437-450.
  26. Doytchinova, I.A., Flower, D.R., VaxiJen: a server for prediction of protective antigens, tumour antigens and subunit vaccines. BMC Bioinformatics, 2007, Vol. 8, p. 4.
  27. EL‐Manzalawy, Y., Dobbs, D., Honavar, V., Predicting linear B‐cell epitopes using string kernels. J. Mol. Recogn, 2008, Vol. 21, pp. 243–55.
  28. Kringelum, J.V., Lundegaard, C., Lund, O., Nielsen, M., Reliable B cell epitope predictions: impacts of method development and improved benchmarking. PLoS Comput Biol, 2012, p. e1002829.
  29. Ansari, H.R., Raghava, G.P., Identification of conformational B-cell Epitopes in an antigen from its primary sequence. Immunome Res, 2010, Vol. 6, p. 6.
  30. Guan, P., Doytchinova, I.A., Zygouri, C., Flower, D.R., MHCPred: A server for quantitative prediction of peptide-MHC binding. Nucleic Acids Res, 2003, Vol. 31, pp. 3621–3624.
  31. Bhasin, M., Raghava, G.P.S., A hybrid approach for predicting promiscuous MHC class I restricted T cell epitopes.J. Biosci, 2007, Vol. 32, pp. 31–42.
  32. Zhang, Q., Wang, P., Kim, Y., Haste-Andersen, P., Beaver, J., Bourne, P.E., Bui, H.H., Buus, S., Frankild, S., Greenbaum, J., Lund, O., Immune epitope database analysis resource (IEDB-AR). Nucleic Acids Res, 2008, Vol. 36, pp. 513-518.
  33. Saha, S., Raghava, G.P.S., AlgPred: prediction of allergenic proteins and mapping of IgE epitopes. Nucleic Acids Res, 2006, Vol. 34, pp. 202–209.
  34. Dimitrov, I., Bangov, I., Flower, D.R., Doytchinova, I., AllerTOP v.2—a server for in silico prediction of allergens. J Mol Model, 2014, Vol. 20, p. 2278.
  35. Mohammad, N., Karsabet, M.T., Amani, J., Ardjmand, A., Zadeh, M.R., Gholi, M.K., Saffari, M. and Ghasemi, A., In silico design of a chimeric protein containing antigenic fragments of Helicobacter pylori; a bioinformatic approach. Open Bioinforma, 2016, Vol. 10, p. 97.
  36. Amani, J., Salmanian, A.H., Rafati, S., Mousavi, S.L., Immunogenic properties of chimeric protein from espA, eae and tir genes of Escherichia coli O157: H7. Vaccine, 2010, Vol. 28, pp. 6923-6929.
  37. Arai, R., Ueda, H., Kitayama, A., Kamiya, N., Nagamune, T., Design of the linkers which effectively separate domains of a bifunctional fusion protein. Protein Eng Des Sel, 2001, Vol. 14, pp. 529-532.
  38. Arai, R., Wriggers, W., Nishikawa, Y., Nagamune, T., Fujisawa, T., Conformations of variably linked chimeric proteins evaluated by synchrotron X‐ray small‐angle scattering. Proteins, 2004, Vol. 57, pp. 829-838.
  39. Webb, B., Sali, A.,  Comparative protein structure modeling using Modeller. Curr Protoc Bioinformatics, 2014, Vol. 47, pp.1-32.
  40. Greenbaum, J., Sidney, J., Chung, J., Brander, C., Peters, B., Sette, A., Functional classification of class II human leukocyte antigen (HLA) molecules reveals seven different supertypes and a surprising degree of repertoire sharing across supertypes. Immunogenetics, 2011, Vol. 63, pp. 325–335.
  41. Bakhshi, M., Ebrahimi, F., Zargan, J., Nazarian, S., Sheikhzade, V., Cloning and Recombinant Expression of EspA as a Virulence Factor of E. coli O157: H7. JMUMS, 2014, Vol. 24, pp. 12-20.
  42. Rabinovitz, B.C., Larzábal, M., Vilte, D.A., Cataldi, A., Mercado, E.C., The intranasal vaccination of pregnant dams with Intimin and EspB confers protection in neonatal mice from Escherichia coli (EHEC) O157: H7 infection. Vaccine, 2016, Vol. 34, pp. 2793-2797.
  43. Yazdanparast, A., Mousavi, S.L., Rasooli, I., Amani, J., Jalalinadoushan, M., Immunogenical Study of Chimeric Recombinant Intimin-Tir of Escherichia coli O157: H7 in Mice.Arch Clin, 2012, Vol. 7, pp. 45-51.
  44. Novinrooz, A., Salehi, T.Z., Firouzi, R., Arabshahi, S., Derakhshandeh, A., In-silico design, expression, and purification of novel chimeric Escherichia coli O157: H7 OmpA fused to LTB protein in Escherichia coli. PLoS One, 2017, Vol. 12, pp. e 0173761.