Abstract
Bioenergetics is one of the oldest fields in microbiology research, yet the intricacies of these pathways in bacterial physiology remains poorly understood, especially outside of model organisms. Targeting bacterial metabolism is being increasingly recognized as a promising strategy in the fight against antimicrobial resistance (AMR); as by disrupting core energy pathways, the survival, adaptation or tolerance of a bacterium can be influenced. One metabolic pathway of growing interest is the electron transport chain (ETC) due to its roles in energy production, aerobic adaptation, and reactive oxygen species (ROS) generation. Several ETC components have already been identified as antimicrobial targets in Mycobacterium tuberculosis, and Staphylococcus aureus. This research focused on Enterococcus faecalis, a common hospital-associated pathogen with rising AMR. When provided with exogenous hemin, E. faecalis can assemble a simple but functional ETC consisting of a type II NADH:quinone oxidoreductase (Ndh2), demethylmenaquinone, and a cytochrome bd oxidase (CydAB). Additionally, its F-type ATPase/synthase can either generate ATP using a protonmotive force or hydrolyse ATP to maintain this gradient. Understanding these energy systems can reveal metabolic weak points in stress-tolerant organisms such as E. faecalis.
In this work, the aerobic respiratory components were assessed with respect to the metabolism and energetics of E. faecalis across different nutrient conditions and oxygen levels. Metabolic profiling, oxygen consumption assays, and growth kinetics were used to identify a non- essential but significant shift in the metabolomes of E. faecalis with the loss of cydAB or the alternative cytosolic NADH oxidase. Protocols to quantify the protonmotive force were established and consequently used to establish the reverse activity of an F-type ATPase/synthase is the primary generator of proton gradient. Furthermore, the native membrane potential of E. faecalis was classified as intrinsically low which points to the proton gradient as the dominant mechanism of the PMF. Finally, the effects of the respiratory chain on antimicrobial susceptibility were explored. A novel vulnerability in NADH homeostasis was identified, which translated to increased sensitivity to redox-cycling antimicrobial clofazimine in E. faecalis. Fluorescence microscopy techniques were developed to monitor ROS accumulation, and show remarkable robustness to ROS damage. These techniques elucidated a remarkable tolerance to membrane depolarization in E. faecalis, which may explain its resilience to antibiotics like daptomycin. This work highlights the importance of bioenergetics in understanding and targeting metabolic resilience in clinically relevant bacteria.