Abstract
Antimicrobial tolerance is the ability of a bacterium to survive over long periods of time in the presence of high antimicrobial concentrations and is an important precursor in the development of antimicrobial resistance. Despite this, the molecular mechanisms underpinning tolerance remain largely unknown. Previous work from our group identified the two-component regulatory system CroRS as a mediator of tolerance to teixobactin (TXB) in Enterococcus faecalis. The aim of this study was to identify the TXB-induced CroRS regulon in E. faecalis and understand the mechanisms of TXB cell killing in the wild-type and ∆croRS strain.
Through transcriptomic analyses, we have identified the CroRS-regulated TXB-induced regulon to comprise 132 genes (>1 Log2 fold-change). Importantly, hypergeometric testing identified significant enrichment of genes involved in lipid, polyketide, and terpenoid metabolism in the CroRS regulon (P < 0.05). We rescued tolerance in the croRS deletion strain by passaging a DcroRS strain for wild-type growth in the absence of antimicrobials and observed numerous mutations in a heptaprenyl diphosphate synthase (hppS, EF2057), a component of the quinone biosynthesis pathway. We hypothesise that this allows rerouting of the terpenoid end product farnesyl diphosphate from quinone biosynthesis to cell wall biogenesis, rescuing tolerance in the DcroRS strain. Furthermore, the hppS mutant in the DcroRS background displays an increased tolerance to antimicrobial-induced killing and cell-damaging agents. Overall, this research has identified the antimicrobial-induced CroRS regulon and isolated a critical pathway in antimicrobial tolerance.
Previous studies have identified that TXB kills Staphylococcus aureus through a dysregulation of cell wall autolysins, resulting in cell lysis and death. The mechanism of cell death in E. faecalis remains unknown. Here, the two major mechanisms of bactericidal antimicrobial killing, that is, cell death via dysregulation of cell wall autolysins and death via an increase in ROS, were investigated to determine how CroRS protects from TXB killing in E. faecalis. Autolysis assays revealed that TXB exerts an autolytic-dependent mechanism of action in DcroRS, indicating that CroRS protects against TXB-induced autolysis. Importantly, we uncovered that TXB treatment generated more reactive oxygen species (e.g. O2- and H2O2) in the DcroRS strain compared to the wild-type, which suggests that CroRS plays a crucial role in protecting against TXB-induced reactive oxygen species. Taken together, these findings suggest that TXB has a novel dual mechanism of killing E. faecalis, with CroRS being crucial for protection against TXB-induced ROS and TXB-indued autolysis.
We identified and characterised two compounds, gambogic acid and ursonic acid, that effectively synergised with sub-lethal concentrations of vancomycin to kill E. faecalis, including a vancomycin-resistant strain. The molecular target of these compounds in E. faecalis remain unknown, but mutants resistant to these compounds suggested a role for a two component regulatory system for gambogic acid resistance and the terpenoid pathway for ursonic acid resistance in the DcroRS strain.
Overall, this thesis provides fundamental insights into the role of CroRS in TXB tolerance in E. faecalis. In addition, it has identified a novel strategy to overcoming VRE via synergy with natural products as a potential therapy for treatment of resistant E. faecalis.