Exploring Daptomycin Hypersensitivity in : The Impact of LafB Mutation on Bacterial Virulence.

Daptomycin (DAP) is a therapeutic option for vancomycin-resistant (VRE) infections, but DAP resistance may occur during treatment. Previously, we identified a mutation within the gene that induces hypersusceptibility to DAP. The gene encodes a glycosyltransferase involved in lipoteichoic acid anchor synthesis, which makes it a promising target for enhancing DAP efficacy. In this study, we characterized LafB protein (LafB) biophysical properties, used AlphaFold3 to predict LafB in silico three-dimensional structure, and determined gene mutation's role in virulence, comparing HBSJRP18 (DAP-hypersusceptible) and a revertant, HBSJRP18_2.7, and analyzing bacterial growth kinetics, biofilm formation ability, and virulence in a model. After gene cloning and expressing and purifying LafB, circular dichroism and SEC-MALS assays revealed its monomeric nature under in vitro conditions, with approximately a 40 kDa molecular mass and a melting temperature of 50 °C. In silico prediction indicated that LafB is an αβ-type protein with two domains conforming to the GT-4 family glycosyltransferases. These results are further supported by the highly conserved amino acids (E257, D91, R184, and K185), likely involved in UDP-Glc binding. The studied gene mutation resulted in a significant decrease in bacterial growth and virulence in the invertebrate model.