Metabolomics of Escherichia coli Treated with the Antimicrobial Carbon Monoxide-Releasing Molecule CORM-3 Reveals Tricarboxylic Acid Cycle as Major Target

Abstract

In the last decade, carbon monoxide-releasing molecules (CORMs) have been shown to act against several pathogens and to be promising antimicrobials. However, the understanding of the mode of action and reactivity of these compounds on bacterial cells is still deficient. In this work, we used a metabolomics approach to probe the toxicity of the ruthenium(II) complex Ru(CO)3Cl(glycinate) (CORM-3) on Escherichia coli. By resorting to 1H nuclear magnetic resonance, mass spectrometry, and enzymatic activities, we show that CORM-3-treated E. coli accumulates larger amounts of glycolytic intermediates, independently of the oxygen growth conditions. The work provides several evidences that CORM-3 inhibits glutamate synthesis and the iron-sulfur enzymes of the tricarboxylic acid (TCA) cycle and that the glycolysis pathway is triggered in order to establish an energy and redox homeostasis balance. Accordingly, supplementation of the growth medium with fumarate, α-ketoglutarate, glutamate, and amino acids cancels the toxicity of CORM-3. Importantly, inhibition of the iron-sulfur enzymes glutamate synthase, aconitase, and fumarase is only observed for compounds that liberate carbon monoxide. Altogether, this work reveals that the antimicrobial action of CORM-3 results from intracellular glutamate deficiency and inhibition of nitrogen and TCA cycles.

‘Carbon-Monoxide-Releasing Molecule-2 (CORM-2)’ Is a Misnomer: Ruthenium Toxicity, Not CO Release, Accounts for Its Antimicrobial Effects

“…Other investigators have suggested that antimicrobial activity is due in part to generation of reactive oxygen species, perhaps following respiratory inhibition [46,47]. Recently, a further proposal for CORM-3 toxicity in bacteria has been advanced [48], namely that it elicits intracellular glutamate deficiency…”

Photo-activated CO-release in the amino tungsten Fischer carbene complex, [(CO)5WC(NC4H8)Me], picosecond time resolved infrared spectroscopy, time-dependent density functional theory, and an antimicrobial study

“…CO trapping studies indicate that CO is predominantly responsible for the bactericidal activity. While carbon monoxide-releasing molecules have been shown to act as promising antimicrobials against several pathogens over the last decade [39,73,74], the mode of action is not fully clear and depending on the compound, multiple interactions with intracellular targets may occur that result in cell membrane perturbations, inhibition of DNA repair or iron chelation. Determining the fate of CO and its mechanisms of interaction with bacteria was beyond the scope of this work.…”

Hydrogen Sulfide and Carbon Monoxide Tolerance in Bacteria

“…Still, CO also targets non-heme proteins, as inferred by the similar CO susceptibility of heme-deficient (Δ hemA ) and wild-type strains of E. coli [ 100 ]. A metabolomic study of E. coli treated with the CO releaser CORM-3 reported the impairment of glutamate synthesis and inactivation of iron-sulfur enzymes, such as aconitase and fumarase, causing intracellular glutamate deficiency and inhibition of the nitrogen and TCA cycles [ 101 ]. In strains of sulfate-reducing bacteria of the Desulfovibrio genus, high CO concentrations (20–70% v/v ) inactivated hydrogenase and superoxide dismutase enzymes, and stimulated formation of ROS [ 87 , 88 , 89 ].…”