Related Experiment Video
Updated: Sep 16, 2025
Invasion of Human Cells by a Bacterial Pathogen
Published on: March 21, 2011
Exploring Daptomycin Hypersensitivity in Enterococcus faecium: The Impact of LafB Mutation on Bacterial Virulence
Pamela I Huanambal Esquén1, Diego A Leonardo1, Livia R Manzine1
1Department of Physics and Interdisciplinary Science, Sao Carlos Institute of Physics, University of Sao Paulo, Sao Carlos 13563-120, Brazil.
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 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 lafB gene mutation resulted in a significant decrease in bacterial growth and virulence in the invertebrate model.
Insights
A mutation in the Enterococcus faecium lafB gene enhances susceptibility to daptomycin (DAP). This finding offers a new strategy to combat vancomycin-resistant Enterococcus faecium infections by targeting lipoteichoic acid synthesis.
Area of Science:
- Microbiology
- Biochemistry
- Structural Biology
Background:
- Daptomycin (DAP) is crucial for treating vancomycin-resistant Enterococcus faecium (VRE) infections.
- Daptomycin resistance in VRE can emerge during therapy.
- A previously identified mutation in the E. faecium lafB gene confers hypersusceptibility to DAP.
Purpose of the Study:
- To characterize the biophysical properties of the E. faecium LafB protein (EfLafB).
- To predict the 3D structure of LafB using AlphaFold3.
- To investigate the role of the lafB mutation in bacterial virulence and growth.
Main Methods:
- Protein expression, purification, and biophysical characterization (circular dichroism, SEC-MALS) of EfLafB.
- In silico 3D structure prediction of LafB.
- Comparative analysis of bacterial growth, biofilm formation, and Galleria mellonella virulence between a DAP-hypersusceptible strain and a lafB revertant.
Main Results:
- EfLafB is a monomeric protein with a molecular mass of approximately 40 kDa and a melting temperature of 50 °C.
- In silico analysis predicted LafB as an αβ-type glycosyltransferase (GT-4 family) with conserved residues potentially involved in UDP-Glc binding.
- The lafB mutation significantly reduced bacterial growth and virulence in the Galleria mellonella model.
Conclusions:
- The lafB gene, encoding a glycosyltransferase in lipoteichoic acid synthesis, is a viable target for enhancing DAP efficacy against VRE.
- The characterized biophysical and structural properties of EfLafB provide insights into its function.
- Targeting lafB represents a promising strategy to overcome DAP resistance in VRE infections.
More Related Videos
08:45Whole Genome Sequencing of Candida glabrata for Detection of Markers of Antifungal Drug Resistance
Published on: December 28, 2017
05:06Author Spotlight: Advancing Antibiotic Resistance Research Using an Efflux-Deficient Bacterial Strain and a Single-Copy Gene Expression System
Published on: January 5, 2024
