This video was the winning entry of the FEBS Network Video Competition 2023. Congratulations to Filippo Vascon, from the Department of Biology, University of Padua, in Padua, Italy. Below is a summary of the reseach presented in the video.
Anti-LexA Nanobodies to Fight Antibiotic Resistance
The SOS response is a bacterial pathway activated in reply to DNA damage, a common outcome of antibiotic treatment. The RecA protein recognizes ssDNA and activates itself by oligomerizing on it. The central event in the SOS pathway is the autoproteolysis of the transcriptional repressor LexA, which is induced by activated RecA. LexA-regulated SOS genes are, thus, upregulated and mediate the acquisition of antibiotic resistance and virulent phenotypes.
To suppress this pathway, we developed nanobodies that bind LexA and inhibit its autoproteolytic activity.
A library of nanobodies was obtained by llama immunization, enriched in LexA binders by phage display and screened for LexA inhibitors by a functional assay relying on fluorescence polarization. The fluorescence polarization signal decreases following the RecA-induced cleavage of a fluorescently labeled LexA. This assay was used also to quantify the inhibitory potency of the selected nanobodies (IC50 ~1µM).
For LexA autoproteolysis to happen, its cleavable loop (the substrate of proteolysis) needs to enter the active pocket in which the catalytic dyad resides, and this movement is triggered by activated RecA. Our nanobodies were shown by x-ray crystallography to bind LexA cleavable loop and trap it in an inactive conformation, far away from the active site.