Insights into the Antibacterial Mechanism of Action of Chelating Agents by Selective Deprivation of Iron, Manganese, and Zinc

Abstract

Bacterial growth and proliferation can be restricted by limiting the availability of metal ions in their environment. Humans sequester iron, manganese and zinc to help prevent infection by pathogens, a system termed nutritional immunity. Commercially-used chelants have high binding affinities with a variety of metal ions, which may lead to antibacterial properties that mimic these innate immune processes. However, the modes of action of many of these chelating agents in bacterial growth inhibition and their selectivity in metal deprivation in cellulo remain ill-defined. We address this shortcoming by examining the effect of eleven chelators on Escherichia coli growth and their impact on the cellular concentration of five metals. Four distinct effects were uncovered: i) no apparent alteration in metal composition, ii) depletion of manganese alongside reductions in iron and zinc levels, iii) reduced zinc levels with a modest reduction in manganese, and iv) reduced iron levels coupled with elevated manganese. These effects do not correlate with the absolute known chelant metal ion affinities in solution, however, for at least five chelators for which key data are available, they can be explained by differences in the relative affinity of chelants for each metal ion. The results reveal significant insights into the mechanism of growth inhibition by chelants, highlighting their potential as antibacterials and as tools to probe how bacteria tolerate selective metal deprivation. IMPORTANCE Chelating agents are widely used in industry and consumer goods to control metal availability, with bacterial growth restriction as a secondary benefit for preservation. However, the antibacterial mechanism of action of chelants is largely unknown, particularly with respect to the impact on cellular metal concentrations. The work presented here uncovers distinct metal starvation effects imposed by different chelants on the model Gram-negative bacterium Escherichia coli . The chelators were studied both individually and in pairs with the majority producing synergistic effects in combinations that maximise antibacterial hostility. The judicious selection of chelants based on contrasting cellular effects should enable reductions in the quantities of chelant required in numerous commercial products and presents opportunities to replace problematic chemistries with biodegradable alternatives.

A critical role for iron and zinc homeostatic systems in the evolutionary adaptation of Escherichia coli to metal restriction

“…Finally, the observed resistance mechanisms fit with the known effect of the two chelants on cellular metal levels [16]. Since growth in the presence of EDTA can be restored by improving zinc distribution, this metal would seem to be primarily responsible for growth inhibition by EDTA.…”

“…coli fitness. Should evolvable resistance prove problematic, it could be overcome either by utilizing chelator pairs with contrasting metal selectivities [16] or in combinations with other antimicrobial agents [21, 68–71].…”

“…However, the results provided here are encouraging and suggest that disrupting the finely tuned balance of metal homeostatic systems also has detrimental consequences for E. coli fitness. Should evolvable resistance prove problematic, it could be overcome either by utilizing chelator pairs with contrasting metal selectivities [16] or in combinations with other antimicrobial agents [21,[68][69][70][71].…”

Virulence regulation of Zn2+ uptake system znuABC on mesophilic Aeromonas salmonicida SRW-OG1

“…Among all the essential trace elements in living organisms, iron (Fe) is the first abundant transition metal element, followed by zinc (Zn) ( 14 – 16 ). Zn is present in the vast majority of proteins.…”

Effect of the zinc transporter ZupT on the virulence mechanisms of mesophilic Aeromonas salmonicida SRW‐OG1

“…Transition metal elements, such as iron (Fe) and zinc (Zn), are highly demanded by living organisms [1,[19][20][21]. In prokaryotes, approximately 10% of proteins require zinc for their formation, including zinc-binding proteins [1,[22][23][24].…”