Mechanisms of bactericidal action and β-lactam synergy by the zinc ionophore PBT2

Abstract

The emergence and global spread of antimicrobial resistance (AMR) is an increasingly serious threat to human and veterinary medicine. Driven by the misuse and overuse of antibiotics, resistance to our antibiotic armoury is rapidly outpacing our current efforts to discover and develop novel agents. In light of this, there is restimulated interest in exploiting metals for novel antibacterial strategies. Zinc is an essential metal ion for various cellular pathways in bacterial physiology, yet excess amounts are toxic. The involvement of zinc in modulating antibiotic efficacy and resistance pathways is also becoming increasingly evident. Zinc ionophores, molecules that transport zinc into cells, offer a strategy to capitalize on the anti-infective potential of this metal. The zinc ionophore PBT2, an 8-hydroxyquinoline derivative, has recently been discovered to potentiate zinc toxicity and antibiotic efficacy against several AMR pathogens, suggesting possibilities for zinc ionophores as a novel class of standalone antibiotic or antibacterial adjuvant in either animal or human medicine. A molecular understanding of these actions, however, is currently lacking. This body of work examines the bactericidal action of PBT2 against the veterinary pathogen Streptococcus uberis, and the underlying mechanism of synergy between PBT2 and β-lactam antibiotics against methicillin-resistant Staphylococcus aureus (MRSA). A combination of bacterial physiology, biochemistry, genetics, and chemical biology approaches were utilised to investigate the mechanisms of zinc ionophores in both lines of this research. We revealed PBT2-mediated intracellular zinc accumulation disrupts zinc and manganese homeostasis and cellular redox balance in S. uberis, ultimately exerting its bactericidal action through intracellular zinc accumulation and manganese starvation, ROS accumulation, and the impairment of manganese-dependent antioxidant activity. We further demonstrated that β-lactam antibiotic resensitization in MRSA by PBT2 and related structural analogues is likewise associated with a destabilization of cellular zinc and manganese homeostasis. Mechanistic investigations revealed this zinc ionophoric activity perturbs multiple elements involved in β-lactam resistance in MRSA, including the expression of key resistance determinants, cell wall and membrane integrity, and the proton motive force (PMF). Further, PBT2 and β-lactam combination treatment significantly improved animal survival rates and pathogen clearance in a murine model of invasive MRSA infection. This study provides novel mechanisms of intracellular zinc toxicity and demonstrates a targetable overlap between metal homeostasis, cell wall and membrane metabolism and β-lactam resistance. Overall, this data contributes and adds to the field of bacterial metal homeostasis and exemplifies how it can be exploited for novel antibacterial strategies in veterinary and human medicine.