Structural Basis for the Specific Neutralization of Stx2a with a Camelid Single Domain Antibody Fragment.

Robert Bernedo-Navarro,Henri De Greve,Ema Romão,Yann Sterckx,Tomomasa Yano,Joar Pinto,Serge Muyldermans

doi:10.3390/toxins10030108

Abstract

Background: Shiga toxin-producing Escherichia coli (STEC) are a subset of pathogens leading to illnesses such as diarrhea, hemolytic uremic syndrome and even death. The Shiga toxins are the main virulence factors and divided in two groups: Stx1 and Stx2, of which the latter is more frequently associated with severe pathologies in humans. Results: An immune library of nanobodies (Nbs) was constructed after immunizing an alpaca with recombinant Shiga toxin-2a B subunit (rStx2aB), to retrieve multiple rStx2aB-specific Nbs. The specificity of five Nbs towards rStx2aB was confirmed in ELISA and Western blot. Nb113 had the highest affinity (9.6 nM) and its bivalent construct exhibited a 100-fold higher functional affinity. The structure of the Nb113 in complex with rStx2aB was determined via X-ray crystallography. The crystal structure of the Nb113–rStx2aB complex revealed that five copies of Nb113 bind to the rStx2aB pentamer and that the Nb113 epitope overlaps with the Gb3 binding site, thereby providing a structural basis for the neutralization of Stx2a by Nb113 that was observed on Vero cells. Finally, the tandem-repeated, bivalent Nb1132 exhibits a higher toxin neutralization capacity compared to monovalent Nb113. Conclusions: The Nb of highest affinity for rStx2aB is also the best Stx2a and Stx2c toxin neutralizing Nb, especially in a bivalent format. This lead Nb neutralizes Stx2a by competing for the Gb3 receptor. The fusion of the bivalent Nb1132 with a serum albumin specific Nb is expected to combine high toxin neutralization potential with prolonged blood circulation.

Highlights

Shiga toxin-producing Escherichia coli (STEC) is a heterogeneous group of microorganisms, which causes around three million cases of acute illnesses in humans each year [1]
We describe the identification and characterization of a nanobody (Nb113) with the potential to neutralize the Stx2a and Stx2c toxins that are associated with human clinical infections [6,7,8]
It is well established that the Shiga toxins (Stx) B5 domain, even in absence of the Stx A subunit, forms a pentameric architecture that is functionally equivalent to the Stx holotoxin in terms of Gb3 receptor binding [27]

Summary

Introduction

Shiga toxin-producing Escherichia coli (STEC) is a heterogeneous group of microorganisms, which causes around three million cases of (potentially fatal) acute illnesses in humans each year [1].A subset of STEC, the enterohemorrhagic E. coli (EHEC) pathotype, is comprised of strains that are typically associated with illnesses in humans. Shiga toxin-producing Escherichia coli (STEC) is a heterogeneous group of microorganisms, which causes around three million cases of (potentially fatal) acute illnesses in humans each year [1]. The average cost per STEC case varies greatly according to the severity of the illness in patients. It ranges from less than $30 for those that do not require medical care to more than $6 million for patients with HUS with eventually a fatal outcome [3,4]. Shiga toxin-producing Escherichia coli (STEC) are a subset of pathogens leading to illnesses such as diarrhea, hemolytic uremic syndrome and even death. Conclusions: The Nb of highest affinity for rStx2aB is the best Stx2a and Stx2c toxin neutralizing Nb, especially in a bivalent format

Methods

Results

Conclusion