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).
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.