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Development and characterization of recombinant antibody fragments that recognize and neutralize in vitro Stx2 toxin from Shiga toxin-producing Escherichia coli.

Luz D, Chen G, Maranhão AQ, Rocha LB, Sidhu S, Piazza RM - PLoS ONE (2015)

Bottom Line: Stx toxin is a member of the AB5 family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane.Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans.In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Bacteriologia, Instituto Butantan, São Paulo, Brazil.

ABSTRACT

Background: Stx toxin is a member of the AB5 family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane. Shiga toxin-producing Escherichia coli strains (STEC) may produce Stx1 and/or Stx2 and variants. Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. The development of tools for the detection and/or neutralization of these toxins is a turning point for early diagnosis and therapeutics. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes.

Methods and findings: In this work, we developed two recombinant antibodies; scFv fragments from mouse hybridomas and Fab fragments by phage display technology using a human synthetic antibody library. Both fragments showed high binding affinity to Stx2, and they were able to bind specifically to the GKIEFSKYNEDDTF region of the Stx2 B subunit and to neutralize in vitro the cytotoxicity of the toxin up to 80%. Furthermore, the scFv fragments showed 79% sensitivity and 100% specificity in detecting STEC strains by ELISA.

Conclusion: In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro.

No MeSH data available.


Related in: MedlinePlus

Structural analyses of antibody fragments.A. Peptide mapping and the corresponding peptides, with the epitopes highlighted in pink. B. Structure prediction of both recombinant antibodies and Stx2, subunit A (purple) and subunit B (green), with the recognized peptides highlighted in pink as well as the antibody CDRs of recombinant antibodies. Also, the variable chains are represented, heavy (blue) and light (purple). In the Fab structure, the constant chain is shown in light blue, as well as the scFv linker.
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pone.0120481.g003: Structural analyses of antibody fragments.A. Peptide mapping and the corresponding peptides, with the epitopes highlighted in pink. B. Structure prediction of both recombinant antibodies and Stx2, subunit A (purple) and subunit B (green), with the recognized peptides highlighted in pink as well as the antibody CDRs of recombinant antibodies. Also, the variable chains are represented, heavy (blue) and light (purple). In the Fab structure, the constant chain is shown in light blue, as well as the scFv linker.

Mentions: The recognition of Stx1 and Stx2 toxins by scFv and Fab prompted us to determine which epitope was involved in antibody binding. Peptide mapping showed that both fragments bound to the same epitope on the B subunit, the toxin subunit responsible for binding to the cell receptor (Fig. 3A). The peptide recognized in the Stx1 B subunit was YTKYNDDTFT (highlighted in pink, Fig. 3B), and for the Stx2 B subunit, the fragments were able to recognize the GKIEFSKYNEDDTF epitope (highlighted in pink, Fig. 3B), which is larger than the epitope in Stx1 toxin. The homologous sequence between B subunits of Stx1 and Stx2 was KYN(E)DDTF, which can explain the cross reactivity of these fragments.


Development and characterization of recombinant antibody fragments that recognize and neutralize in vitro Stx2 toxin from Shiga toxin-producing Escherichia coli.

Luz D, Chen G, Maranhão AQ, Rocha LB, Sidhu S, Piazza RM - PLoS ONE (2015)

Structural analyses of antibody fragments.A. Peptide mapping and the corresponding peptides, with the epitopes highlighted in pink. B. Structure prediction of both recombinant antibodies and Stx2, subunit A (purple) and subunit B (green), with the recognized peptides highlighted in pink as well as the antibody CDRs of recombinant antibodies. Also, the variable chains are represented, heavy (blue) and light (purple). In the Fab structure, the constant chain is shown in light blue, as well as the scFv linker.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4366190&req=5

pone.0120481.g003: Structural analyses of antibody fragments.A. Peptide mapping and the corresponding peptides, with the epitopes highlighted in pink. B. Structure prediction of both recombinant antibodies and Stx2, subunit A (purple) and subunit B (green), with the recognized peptides highlighted in pink as well as the antibody CDRs of recombinant antibodies. Also, the variable chains are represented, heavy (blue) and light (purple). In the Fab structure, the constant chain is shown in light blue, as well as the scFv linker.
Mentions: The recognition of Stx1 and Stx2 toxins by scFv and Fab prompted us to determine which epitope was involved in antibody binding. Peptide mapping showed that both fragments bound to the same epitope on the B subunit, the toxin subunit responsible for binding to the cell receptor (Fig. 3A). The peptide recognized in the Stx1 B subunit was YTKYNDDTFT (highlighted in pink, Fig. 3B), and for the Stx2 B subunit, the fragments were able to recognize the GKIEFSKYNEDDTF epitope (highlighted in pink, Fig. 3B), which is larger than the epitope in Stx1 toxin. The homologous sequence between B subunits of Stx1 and Stx2 was KYN(E)DDTF, which can explain the cross reactivity of these fragments.

Bottom Line: Stx toxin is a member of the AB5 family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane.Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans.In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro.

View Article: PubMed Central - PubMed

Affiliation: Laboratório de Bacteriologia, Instituto Butantan, São Paulo, Brazil.

ABSTRACT

Background: Stx toxin is a member of the AB5 family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane. Shiga toxin-producing Escherichia coli strains (STEC) may produce Stx1 and/or Stx2 and variants. Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. The development of tools for the detection and/or neutralization of these toxins is a turning point for early diagnosis and therapeutics. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes.

Methods and findings: In this work, we developed two recombinant antibodies; scFv fragments from mouse hybridomas and Fab fragments by phage display technology using a human synthetic antibody library. Both fragments showed high binding affinity to Stx2, and they were able to bind specifically to the GKIEFSKYNEDDTF region of the Stx2 B subunit and to neutralize in vitro the cytotoxicity of the toxin up to 80%. Furthermore, the scFv fragments showed 79% sensitivity and 100% specificity in detecting STEC strains by ELISA.

Conclusion: In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro.

No MeSH data available.


Related in: MedlinePlus