Abstract
Methicillin-resistant Staphylococcus aureus (MRSA) remains a critical threat to public health due to its remarkable adaptability across diverse host environments and increasing resistance to front-line antibiotics, such as vancomycin and ceftaroline. In response to bacterial invasion, the host immune system utilises a process known as nutritional immunity, involving metal ion intoxication, starvation, and carbon source restriction. Concurrently, phagocytic immune cells generate oxidative stress by producing reactive oxygen species (ROS). However, MRSA and other successful pathogens have evolved sophisticated strategies to counteract these immune pressures, enabling their persistence and proliferation within the host. Understanding how MRSA responds and withstands host-imposed stresses is crucial for developing innovative therapeutic strategies to combat rising antibiotic resistance.
This thesis explores the antibacterial mechanism of the zinc (Zn) ionophore PBT2, which was initially developed to treat neurodegenerative diseases and has emerged as a promising candidate for combating multi-drug resistant (MDR) bacterial pathogens. When PBT2 is administered with Zn (PZ), in vitro, it exhibits potent bactericidal activity, and this combination, when used sub-therapeutically, in vitro and in vivo can restore the efficacy of antibiotics compromised by resistance against Gram-positive and Gram-negative bacteria. PBT2 has been shown to induce toxic Zn accumulation within bacteria, and often a concurrent depletion of Mn and/or Fe. However, a detailed understanding of the cellular targets of metal disruption in response to PBT2 is currently lacking. Our study focuses on MRSA strain USA300 and employs an integrative approach combining transcriptomics, metabolomics, molecular biology, and physiological assays to dissect the molecular consequences of metal ion homeostasis disruption induced by PZ.
We demonstrate that dual metal stress – comprising intracellular Zn toxicity and manganese (Mn) depletion – is essential to PZ’s antibacterial mechanism and ability to resensitise USA300 to the antibiotic oxacillin. Furthermore, we demonstrate that exogenous supplementation with Mn2+ (5 mM) or pyruvate (0.5%) rescues bacterial growth under PZ treatment, emphasising the critical roles of metal starvation and central metabolic flux. PZ triggered widespread metabolic perturbations within central carbon metabolism and associated biosynthetic pathways, including cell wall, amino acid, lipid and nucleotide synthesis. These disruptions destabilised cellular redox balance and led to severe ATP depletion, exposing critical metabolic vulnerabilities in MRSA’s response to metal stress. Importantly, we propose that PZ mimics host immune strategies, such as calprotectin-mediated metal sequestration and macrophage-driven Zn toxicity, but it directly targets intracellular metal pools in an amplified and sustained manner. Further, PBT2 circumvents MRSA’s high-affinity metal transport systems, limits ROS detoxification via Mn-dependent antioxidants, and restricts metabolic flexibility, ultimately leading to cell death. Our findings reveal the clinical potential of PZ, as an adjuvant that boosts the efficacy of various antibiotic classes, and as a standalone agent that may synergise with host immune defenses.
This work highlights a targetable interplay between bacterial metal ion homeostasis, central metabolism, cell envelope integrity and b-lactam resistance. By uncovering how PBT2 subverts MRSA’s adaptive responses, this thesis contributes to our understanding of host-pathogen interactions and offers a foundation for developing novel antimicrobials based on metal homeostasis disruption. Collectively, these findings indicate that PBT2 and related compounds hold promise as sustainable, multifaceted treatments against MRSA and other MDR pathogens, addressing the key challenges of immune evasion and antibiotic resistance.