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

PloS One
Daniela E LuzRoxane M F Piazza

Abstract

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. 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,...Continue Reading

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Citations

Feb 24, 2016·Toxicon : Official Journal of the International Society on Toxinology·Robert Alvin Bernedo-Navarro, Tomomasa Yano
Jan 15, 2019·Brazilian Journal of Microbiology : [publication of the Brazilian Society for Microbiology]·M J Rodrigues-JesusLuís C S Ferreira
May 21, 2016·Proteomics. Clinical Applications·Philipp KuhnMichael Hust
May 22, 2020·Frontiers in Cellular and Infection Microbiology·Sabrina Mühlen, Petra Dersch
Nov 22, 2016·Brazilian Journal of Microbiology : [publication of the Brazilian Society for Microbiology]·Tânia A T GomesMarina B Martinez
Apr 29, 2020·International Journal of Molecular Sciences·Sebastian W MeisterJohn Löfblom
Jul 27, 2021·Frontiers in Cellular and Infection Microbiology·Kristian Daniel Ralph RothMichael Hust
Sep 1, 2021·Immunotherapy·Srichandrasekar Thuthikkadu IndhuprakashThirumalai Diraviyam

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Methods Mentioned

BETA
phage display
ELISA
PCR
Peptide Array

Software Mentioned

GraphPrism
PyMol
GeneScript
Phyre

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