Regulation of Antimicrobial Resistance in Enterococcus faecalis

Abstract

The inappropriate use and overuse of antimicrobials in both clinical and agricultural settings, has led to the rapid emergence and spread of antimicrobial resistance (AMR). Key to combatting AMR will be obtaining a detailed molecular understanding of resistance mechanisms and increasing our knowledge of how bactericidal antimicrobials kill bacteria (pathways of cell death). In this thesis, I have set out to address both of these key issues by understanding how bacitracin is sensed by the opportunistic pathogen Enterococcus faecalis, and how this bacterium responds to the recently discovered antimicrobial teixobactin.

Bacitracin is an antimicrobial peptide that targets the essential lipid carrier undecaprenyl pyrophosphate (UPP) in cell wall synthesis. Several bacitracin resistance mechanisms have been described and high-level resistance is mediated by the BcrRABD resistance cassette. BcrR is a unique membrane-bound DNA-binding one-component regulator which directly senses and responds to bacitracin, initiating the expression of the bcrABD (heterodimeric ABC transporter) resistance operon. Using chemical mutagenesis, we identified key residues involved in bacitracin-binding, dimerisation, and DNA-binding activity to advance our knowledge of how this important regulator functions.

Teixobactin was discovered in 2015 and is an effective bactericidal antimicrobial against important pathogens, including Mycobacterium tuberculosis, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant enterococci. Teixobactin targets pyrophosphate-sugar (PP-sugar) moieties in cell wall biosynthesis and no resistance has been reported thus far. Based on bacitracin and teixobactin target selectivity, we hypothesized that bacitracin resistance mechanisms may confer resistance/tolerance to teixobactin. We found that no reported bacitracin resistance mechanisms including the BcrRABD cassette, a network of two ABC transporters (EF2050-49; EF2752-51), and a two-component regulatory system (EF0926-27), and a BacA-type undecaprenyl pyrophosphate phosphatase (EF2439), that is responsible for the recycling of UPP back to its active state UP during cell wall synthesis, conferred resistance to teixobactin. Whole genome transcriptional profiling of cells exposed to sub-lethal levels of teixobactin identified a peptidoglycan (PG) recycling pathway that was upregulated in response to teixobactin. We hypothesise teixobactin induces an accumulation of muropeptides that act as signalling molecules for the cell wall stress response, mediated through the membrane-bound serine/threonine kinase IreK, and as substrates for PG biosynthesis.