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. 2009 Dec 8:6:28.
doi: 10.1186/1742-4682-6-28.

In silico analysis of chimeric espA, eae and tir fragments of Escherichia coli O157:H7 for oral immunogenic applications

Jafar Amani  1 , S Latif Mousavi, Sima Rafati, Ali H Salmanian
Affiliations

In silico analysis of chimeric espA, eae and tir fragments of Escherichia coli O157:H7 for oral immunogenic applications

Jafar Amani et al. Theor Biol Med Model. .

Abstract

Background: In silico techniques are highly suited for both the discovery of new and development of existing vaccines. Enterohemorrhagic Escherichia coli O157:H7 (EHEC) exhibits a pattern of localized adherence to host cells, with the formation of microcolonies, and induces a specific histopathological lesion (attaching/effacing). The genes encoding the products responsible for this phenotype are clustered on a 35-kb pathogenicity island. Among these proteins, Intimin, Tir, and EspA, which are expressed by attaching-effacing genes, are responsible for the attachment to epithelial cell that leads to lesions.

Results: We designed synthetic genes encoding the carboxy-terminal fragment of Intimin, the middle region of Tir and the carboxy-terminal part of EspA. These multi genes were synthesized with codon optimization for a plant host and were fused together by the application of four repeats of five hydrophobic amino acids as linkers. The structure of the synthetic construct gene, its mRNA and deduced protein and their stabilities were analyzed by bioinformatic software. Furthermore, the immunogenicity of this multimeric recombinant protein consisting of three different domains was predicted.

Conclusion: a structural model for a chimeric gene from LEE antigenic determinants of EHEC is presented. It may define accessibility, solubility and immunogenecity.

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Figures

Figure 1
Figure 1
Schematic model which shows the construction of EspA 120, Intimin 282 and Tir 103, bound together by the linkers for expression in plants; these fragments were selected on the basis of the common sequence found in different strains of E. coli O157 H7.
Figure 2
Figure 2
A: Codon usage analysis of wild type and optimized gene for expression in plants. The value of 100 is set for the codon with the highest usage frequency for a given amino acid in the desired expression into plants. This procedure allows us to compare the adaptiveness of different codons relative to each other (relative adaptiveness). Plots represent the relative adaptiveness of a given codon at the indicated codon position. B: GC analysis of wild type and optimized chimeric gene. Plots represent the average GC content, before and after optimization.
Figure 3
Figure 3
Analysis of chimeric EspA-Intimin-Tir protein secondary structure.
Figure 4
Figure 4
Ab initio and comparative modeling was used to predict the tertiary structure of the chimeric protein, EspA-Intimin-Tir. The result was viewed by Rasmol software.
Figure 5
Figure 5
(A) Evaluation of model stability based on a Ramachandran plot and (B) energy minimization.
See this image and copyright information in PMC

References

    1. Van Diemen PM, Dziva F, Abu-Median A, Wallis TS, Bosch H Van den, Dougan G. et al. Subunit vaccines based on intimin and Efa-1 polypeptides induce humoral immunity in cattle but do not protect against intestinal colonisation by enterohaemorrhagic Escherichia coli O157:H7 or O26:H. Vet Immunol Immunopathol. 2007;116(1-2):47–58. doi: 10.1016/j.vetimm.2006.12.009. - DOI - PMC - PubMed
    1. Babiuk S, Asper DJ, Rogan D, Mutwiri GK, Potter AA. Subcutaneous and intranasal immunization with type III secreted proteins can prevent colonization and shedding of Escherichia coli O157:H7 in mice. Microb Pathog. 2008;45(1):7–11. doi: 10.1016/j.micpath.2008.01.005. - DOI - PubMed
    1. Li Y, Frey E, Mackenzie AM, Finlay BB. Human response to Escherichia coli O157:H7 infection: antibodies to secreted virulence factors. Infect Immun. 2000;68(9):5090–5095. doi: 10.1128/IAI.68.9.5090-5095.2000. - DOI - PMC - PubMed
    1. McNeilly TN, Naylor SW, Mahajan A, Mitchell MC, McAteer S, Deane D. et al. Escherichia coli O157:H7 colonization in cattle following systemic and mucosal immunization with purified H7 flagellin. Infect Immun. 2008;76(6):2594–2602. doi: 10.1128/IAI.01452-07. - DOI - PMC - PubMed
    1. Van Donkersgoed J, Hancock D, Rogan D, Potter AA. Escherichia coli O157:H7 vaccine field trial in 9 feedlots in Alberta and Saskatchewan. Can Vet J. 2005;46(8):724–28. - PMC - PubMed

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