Characterisation of chelator-resistant mutants of Escherichia coli and Staphylococcus aureus: impacts on metal and cell wall homeostasis

Abstract

The necessity of metals for bacterial growth and metabolism has been harnessed in mammalian nutritional immunity to combat invading pathogens. Chelators are metal sequestering agents that have antibacterial properties that resemble those employed by the immune system, and may provide an alternative strategy to counteract increasingly drug-resistant microbes. Despite their widespread applications in industry and medicine, the antibacterial mode of action of chelators is poorly understood. Bacterial adaptation to environmental conditions lacking selected metals is also an underexplored subject area. This thesis addresses these shortcomings by characterising mutants of Escherichia coli and Staphylococcus aureus previously isolated in the presence of two chelators with therapeutic potential, ethylenediaminetetraacetic acid (EDTA) and diethylenetriamine pentamethylene phosphonic acid (DTPMP). The chelators differed in their impact on manganese, iron, and zinc in E. coli, but behaved similarly in S. aureus by restricting manganese. Resistance to EDTA in E. coli was achieved by overproduction of YeiR, a zinc-dependent metallochaperone. Contrastingly, in S. aureus gene mutations linked to cell wall metabolism and teichoic acid modification, affecting penicillin-binding protein 2 (PBP2), or eliminating FmtA or VraF activity, were found to be critical for chelator resistance. The mutants were found to possess thicker cell walls and an increased net negative surface charge due to alterations in teichoic acid D-alanine levels. Phenotypic and cellular metal analyses revealed that loss of FmtA or VraF promotes calcium accumulation, and this protects against loss of manganese to counteract the antibacterial activity of both chelators. Collectively, these studies expand our understanding of the mode of action of chelators against E. coli and S. aureus, and reveal the importance of the cell wall for metal homeostasis in Gram-positive bacteria.