Evaluation of In Vitro Protective Effect of Nanobody/ Intrabody against Bacterial Shiga Toxin: Potential Role of ER Retention Signal (KDEL)

Abstract

Hemolytic-Uremic Syndrome (HUS) clinically presents as a triad of hemolytic anemia, thrombocytopenia and acute renal failure. Mostly, it is caused by infection with certain strains of Shiga toxin-producing E. coli. Recently, a novel class of single-domain antibodies (VHH) or “nanobodies” was raised against Shiga toxin (Stx) and was shown to protect Stx-exposed mice. However, Stx is cleared rapidly from the circulation and is generally not detectable at the time of systemic disease onset. We investigated whether anti-Stx nanobodies (Nbs) can be effective in neutralizing Stx intracellularly. Two Nbs with high affinity to the B subunit of Stx2a were selected to investigate their effect against Shiga toxin. The Nb sequence was cloned in-frame into a backbone vector carrying an Endoplasmic Reticulum (ER) localization and retention signals (KDEL), and Green Fluorescence Protein (GFP). Shiga toxin-sensitive cells (hCMEC/D3, and BxPc3) were transfected with the vector, a cytotoxic dose of the toxin was added after 24 hours and cells viability were determined by resazurin assay at different time points. Intracellular ER-localized expression of anti-Stx nanobodies (JFL47, JGL-34) also the ER-backbone vectors without nanobody insert protected the cell lines at all timepoints. Control plasmids of other signaling peptides [mem: signal sequence targeting plasma membrane+GFP as positive control] or unrelated vectors [N1: empty vector, GFP alone as negative control] did not show significant differences to wildtype. While inconclusive regarding the protective effect of anti-Shiga toxin nanobody constructs, a novel and significant finding of this study is the observed intrinsic, highly protective effect of the ERtargeting sequence (SEKDEL/KDEL).