Exploring daptomycin hypersensitivity in Enterococcus faecium: the impact of LafB mutation on bacterial virulence.
ESQUÉN, Pamela Ibeth Huanambal; CABREJOS, Diego Antonio Leonardo; MANZINE, Livia Regina; FARRO, Erick Giancarlo Suclupe; KAJFASZ, Jessica K.; MELLO, Suelen Scarpa de; NOGUEIRA, Mara Corrêa Lelles; MUNIZ, João Renato Carvalho; NASCIMENTO, Alessandro Silva; GILMORE, Michael S.; ABRANCHES, Jacqueline; LEMOS, Jose A.; CAMARGO, Ilana Lopes Baratella da Cunha.
ESQUÉN, Pamela Ibeth Huanambal; CABREJOS, Diego Antonio Leonardo; MANZINE, Livia Regina; FARRO, Erick Giancarlo Suclupe; KAJFASZ, Jessica K.; MELLO, Suelen Scarpa de; NOGUEIRA, Mara Corrêa Lelles; MUNIZ, João Renato Carvalho; NASCIMENTO, Alessandro Silva; GILMORE, Michael S.; ABRANCHES, Jacqueline; LEMOS, Jose A.; CAMARGO, Ilana Lopes Baratella da Cunha.
Abstract: Daptomycin (DAP) is a therapeutic option for vancomycin-resistant Enterococcus faecium (VRE) infections, but DAP resistance may occur during treatment. Previously, we identified a mutation within the E. faecium lafB gene that induces hypersusceptibility to DAP. The lafB 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 E. faecium LafB protein (EfLafB) biophysical properties, used AlphaFold3 to predict LafB in silico three-dimensional structure, and determined lafB gene mutation?s role in virulence, comparing E. faecium HBSJRP18 (DAP-hypersusceptible) and a lafB revertant, HBSJRP18_2.7, and analyzing bacterial growth kinetics, biofilm formation ability, and virulence in a Galleria mellonella model. After gene cloning and expressing and purifying EfLafB, 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 aß-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 lafB gene mutation resulted in a significant decrease in bacterial growth and virulence in the invertebrate model.
@article={003255115,author = {ESQUÉN, Pamela Ibeth Huanambal; CABREJOS, Diego Antonio Leonardo; MANZINE, Livia Regina; FARRO, Erick Giancarlo Suclupe; KAJFASZ, Jessica K.; MELLO, Suelen Scarpa de; NOGUEIRA, Mara Corrêa Lelles; MUNIZ, João Renato Carvalho; NASCIMENTO, Alessandro Silva; GILMORE, Michael S.; ABRANCHES, Jacqueline; LEMOS, Jose A.; CAMARGO, Ilana Lopes Baratella da Cunha.},title={Exploring daptomycin hypersensitivity in Enterococcus faecium: the impact of LafB mutation on bacterial virulence},journal={International Journal of Molecular Sciences},note={v. 26, n. 13, p. 5935-1-5935-17 + supplementary materials},year={2025}}