Fitness costs of vancomycin resistance in Enterococcus

The Gram-positive species Enterococcus faecium and Enterococcus faecalis can be considered prototypical bacterial generalists as they are ubiquitously present as commensals in the guts of humans and animals, while they can also cause infections in immunocompromised individuals. Worryingly, strains of E. faecium and E. faecalis have been able to acquire resistance to the last-resort antibiotic vancomycin. Vancomycin acts by binding to the terminal D-Alanine residue of the peptide stems of peptidoglycan, thus inhibiting the process of the cross-linking of peptidoglycan, which is an essential process in the formation of the bacterial cell wall. Vancomycin resistance gene clusters carried by E. faecium and E. faecalis encode enzymes which replace the terminal D-Ala residue of the peptide stems with D-lactate or D-serine. The two most common mechanisms of vancomycin resistance in both species are vanA- and vanB-type resistance. While these resistance gene clusters are functionally similar, they have evolved independently. While vancomycin-resistant enterococci have become important opportunistic pathogens, their biology has long been understudied, due to a lack of genetic tools that can be used to manipulate enterococcal genomes. In this project, we will leverage recently developed methodologies for genetic manipulation and functional genomics in Enterococcus to accurately quantify the fitness costs of vancomycin resistance in E. faecium and E. faecalis and to determine the mechanisms by which both species minimise these costs. We will generate strains of E. faecium and E. faecalis that are tagged with fluorescent markers (sGFP and mCherry) and which have the vanA and vanB gene clusters inserted in a neutral site on the chromosome. We will use three strains per species, representing different lineages of each species, to generate these constructs in. We will then perform competition assays between the different strains (with or without vancomycin resistance genes), allowing the precise quantification of the fitness costs of vancomycin resistance gene carriage in E. faecium and E. faecalis. Subsequently, we will elucidate how the transcriptional program of E. faecium and E. faecalis is impacted by the presence of vanA- and vanB-type vancomycin resistance genes through high-throughput RNA-sequencing (RNA-seq). We will also quantify the abundance of VanA and VanB on the protein level, to determine whether the data on the mRNA-level aligns with protein levels. To further explore potential differences in the regulation of vancomycin resistance between E. faecium and E. faecalis, we will determine levels of vanA and vanB expression, and the proteins they encode, after a pulse of vancomycin. If mRNA and protein levels remain high, for longer periods of time, after the vancomycin pulse, this will contribute to the energetic cost of resistance. Finally, we will use high-throughput screening of transposon mutant libraries (Tn-seq) to identify genes that contribute to minimising fitness costs of vancomycin resistance in E. faecium and E. faecalis. We will generate targeted deletion mutants in the genes identified by Tn-seq to confirm that they contribute to fitness in the presence of vancomycin and will further characterise the phenotypes of these mutants. This project will provide novel quantitative and mechanistic insights into the trade-offs between antibiotic resistance and its fitness costs in this important group of Gram-positive bacteria. Data generated in this project will open avenues for future studies in Gram-positives on the intersection of antibiotic resistance and bacterial evolution, and may inform the development of novel therapeutics to target vancomycin-resistant enterococci.