Abstract
BackgroundStx 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 FindingsIn 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.ConclusionIn 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.
Highlights
Shiga toxin (Stx)-producing Escherichia coli (STEC) are bacterial pathogens responsible for a spectrum of diseases, ranging from asymptomatic carriage to diarrhea, bloody diarrhea, hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) [1]
We developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for PLOS ONE | DOI:10.1371/journal.pone
Purified Stx1 and Stx2 were purchased from Tufts University School of Medicine, Boston, MA, USA. rProtein A Sepharose Fast Flow was bought from GE Healthcare, UK
Summary
Shiga toxin (Stx)-producing Escherichia coli (STEC) are bacterial pathogens responsible for a spectrum of diseases, ranging from asymptomatic carriage (rare) to diarrhea, bloody diarrhea, hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) [1]. Stx toxins are members of the AB5 family of bacterial toxins, in which the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane and translocate the active A subunit (StxA), which possesses N-glycosidase activity against 28S rRNA of 60S ribosomes into the cytosol, resulting in inhibition of protein synthesis in eukaryotic cells [8,9]. 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 Stx are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes
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