Optimization of the electroporation procedure for OG1RF Enterococcus faecalis to study VanRS_B activation using VanRS_B-GFP reporter construct

Antibiotic-resistant infections have become an increasing threat in modern medicine, since wide-spread antibiotic use in modern medicine. Understanding the resistance mechanisms that evade targeted destruction by antibiotics is vital to developing therapeutics that can counter resistance. Vancomycin is a glycopeptide antibiotic that functions as a cell-wall synthesis inhibitor in Gram-positive bacteria. E. faecalis is a part of the commensal enterococcus genus that inhabits the gastrointestinal tract in humans and animals. Vancomycin inhibits cell wall biosynthesis in E. faecalis. E. faecalis can develop vancomycin-resistance becoming vancomycin-resistant E. faecalis (VRE), with multiple resistance genotypes that are all controlled by a two-component system known as VanR and VanS (VanRS). In Type-B VRE resistance, VRE evades vancomycin-mediated cell death by modifying the normal D-Ala-D-Ala peptidoglycan terminal residue that is susceptible to vancomycin binding and cell-wall synthesis inhibition, to D-Ala-D-Lactate. This alteration prevents the antibiotic from recognizing its binding target, allowing VRE to survive in the presence of vancomycin. When vancomycin is present, VanRS_B expression is upregulated, activating the expression of the downstream resistance genes responsible for facilitating the alteration the cell wall of VRE. We designed a GFP reporter construct in place of the resistance genes in the operon to study the activation mechanism of the Type-B resistance operon. The GFP reporter is under the control of VanR_B and VanS_B. We attempted to place the plasmid into E. faecalis after constructing the reporter-construct, but experienced challenges with the transformation process. Systematic optimization became the focus of this project. We tested different E. faecalis strains, and different concentrations of glycine which weakens the bacterial cell-wall when incorporated, to identify contributing factors that can be adjusted to increase transformation efficiency. We have produced a reporter construct and are currently working on a reproducible electroporation procedure to study vancomycin-induced VanRS_B activation in E. faecalis. Herein I also discuss several contributing factors to transformation failure, experiments to identify specific source(s) preventing transformation success, and future applications such as a dose-response assay to validate function and mutagenesis of VanR_B/S_B or both genes within the reporter-construct both to study vancomycin binding effects.