Antimicrobial Activity of the Manganese Photoactivated Carbon Monoxide-Releasing Molecule [Mn(CO)3(tpa-κ(3)N)](+) Against a Pathogenic Escherichia coli that Causes Urinary Infections

Abstract

Aims: We set out to investigate the antibacterial activity of a new Mn-based photoactivated carbon monoxide-releasing molecule (PhotoCORM, [Mn(CO)3(tpa-κ(3)N)](+)) against an antibiotic-resistant uropathogenic strain (EC958) of Escherichia coli.

Results: Activated PhotoCORM inhibits growth and decreases viability of E. coli EC958, but non-illuminated carbon monoxide-releasing molecule (CORM) is without effect. NADH-supported respiration rates are significantly decreased by activated PhotoCORM, mimicking the effect of dissolved CO gas. CO from the PhotoCORM binds to intracellular targets, namely respiratory oxidases in strain EC958 and a bacterial globin heterologously expressed in strain K-12. However, unlike previously characterized CORMs, the PhotoCORM is not significantly accumulated in cells, as deduced from the cellular manganese content. Activated PhotoCORM reacts avidly with hydrogen peroxide producing hydroxyl radicals; the observed peroxide-enhanced toxicity of the PhotoCORM is ameliorated by thiourea. The PhotoCORM also potentiates the effect of the antibiotic, doxycycline.

Innovation: The present work investigates for the first time the antimicrobial activity of a light-activated PhotoCORM against an antibiotic-resistant pathogen. A comprehensive study of the effects of the PhotoCORM and its derivative molecules upon illumination is performed and mechanisms of toxicity of the activated PhotoCORM are investigated.

Conclusion: The PhotoCORM allows a site-specific and time-controlled release of CO in bacterial cultures and has the potential to provide much needed information on the generality of CORM activities in biology. Understanding the mechanism(s) of activated PhotoCORM toxicity will be key in exploring the potential of this and similar compounds as antimicrobial agents, perhaps in combinatorial therapies with other agents. Antioxid. Redox Signal. 24, 765-780.

FIG. 1.

Structure of PhotoCORM [Mn(CO)₃(tpa-κ³N)]⁺Br⁻. PhotoCORM, photoactivable carbon monoxide-releasing molecule.

FIG. 2.

Detection and quantification of CO release from PhotoCORM using ferrous Mb. A stock solution of Mb (12 μM) was reduced with sodium dithionite. In (A), increasing concentrations of PhotoCORMs were added to myoglobin, followed by exposure to UV light (365 nm) for 6 min, and the difference in absorbance was plotted (globin plus PhotoCORM minus globin, all reduced). Reduced Mb was bubbled with CO gas and plotted as a control. In (B), PhotoCORM (1 mM) was exposed to UV for increasing periods of time while stirring and then added to reduced Mb. Difference spectra were obtained as in (A). Numbers with arrows in the graphs correspond to the concentrations of CO-Mb (μM) formed by the addition of PhotoCORM or CO gas. Plots are representative of three independent repetitions. Mb, myoglobin.

FIG. 3.

Activated PhotoCORM inhibits respiration of EC958-purified membranes and releases CO to oxidases. Isolated membranes from Escherchia coli EC958 were resuspended in Tris-HCl buffer (50 mM, pH 7.4). In (A), are shown representative O₂ electrode traces of O₂ consumption in a closed chamber after adding NADH (arrows) to untreated membranes (control) or membranes exposed to UV light for 6 min in the presence of 200 μM PhotoCORM (PhotoCORM, UV). The Figure also shows 50% and 15% air saturation (dashed lines) at which respiration rates were calculated. In (B), are shown respiration rates at 50% and 15% air saturation from control and PhotoCORM, UV samples [as in (A)], and samples treated with PhotoCORM pre-exposed to UV light for 6 min (pre-illuminated PhotoCORM) or 30 min (to UV light for 30 min with constant stirring) or PhotoCORM kept in the dark (200 μM final concentrations), followed by transfer to the closed chamber. A solution of CO (200 μM) or an equivalent volume of water, followed by exposure to UV light for 6 min, was used as control. Bars represent standard deviation of at least three technical repeats of one representative biological repeat (**p < 0.0001; *p < 0.0005 with respect to the untreated control. In (C), are shown difference spectra (globin plus PhotoCORM or CO minus globin, all reduced) of intact cells of strain EC958 (suspension OD ∼55) treated after reduction with sodium dithionite with either CO gas or 100 μM PhotoCORM. Illumination was for 6–15 min as indicated. OD, optical density.

FIG. 4.

PhotoCORM reduces viability and inhibits aerobic growth of E. coli EC958. Cultures were grown in glucose minimal medium at 37°C, 200 rpm. (A) Shows quantification of CFU from cultures treated with 0 (●), 200 (▲), 350 (▼), and 500 μM PhotoCORMs (■) pre-exposed to UV light for 6 min. In (B), cultures were treated with PhotoCORMs as in (A), but kept in the dark. Growth is represented as CFU/ml of treated cultures divided by the number of CFU/ml at time zero and expressed as log. (C) PhotoCORMs (at 0 [●], 50 [■], 100 [▲], 200 [▼], 300 (♦), and 500 [] μM) were added to cultures, followed by 6 min of exposure to UV light (365 nm). In (D), cultures were treated with PhotoCORMs as in (C), but kept in the dark. (A) and (B) are representative of three independent experiments. Compounds were added at time zero (arrows). Bars represent the standard error of three independent experiments. CFU, colony-forming unit.

FIG. 5.

PhotoCORM releases CO in thick cell suspensions upon exposure to UV. Cell suspensions of E. coli EC958 (OD_600nm = 50) in glucose minimal medium were treated with PhotoCORMs (200 μM) (●) and exposed to UV light for 10 min. Spectra of the headspace were measured every 2 min from 15 min before illumination, during the illumination period, and for 35 min afterward by Fourier transform infrared spectroscopy. For comparison, headspace measurements of PhotoCORM (200 μM) illuminated for 10 min in minimal medium without bacteria were also measured (○). Error bars represent standard deviation of three independent experiments.

FIG. 6.

Intracellular formation of CO-bound bacterial globin from activated PhotoCORM. In (A), E. coli MG1655 cell suspensions overexpressing globin (Ctb) were reduced by addition of glucose (15 mM) and then bubbled with CO gas to saturation or treated with 20 μM PhotoCORMs and exposed to UV light (365 nm) for 6 min. In (B), cell suspensions were treated with increasing concentrations of PhotoCORMs and exposed to UV for 6 min. Difference in absorbance (CO reduced minus reduced) was plotted (numbers indicate PhotoCORM [μM]). In (C), the amount of CO-Ctb formed was calculated from (B) and plotted against PhotoCORM concentration.

FIG. 7.

Combination of activated PhotoCORM with H₂O₂ impairs growth of EC958. Cultures were grown in glucose minimal medium at 37°C, 200 rpm. In (A), cultures were added with 0 (●), 6 (■), 7 (▲), 8 (▼), 9 (♦), and 10 () mM H₂O₂. In (B), control (no additions) (●) and cultures treated with PhotoCORM (100 μM) plus 4 (■), 5 (▲), 6 (▼), 7 (♦), and 8 () mM H₂O₂ were exposed to UV for 6 min. Compounds were added at time zero (arrows). In (C), over the same time scale as in (A) and (B), cell viability is shown in cultures exposed to UV for 6 min in the absence (white bars) or presence of PhotoCORM (100 μM) (light gray bars), H₂O₂ (4 mM) (dark gray bars), or a combination of both compounds (black bars). Samples taken immediately before treatment were recorded as time zero. Bars represent the standard error of at least three independent experiments. Student's test was used to compare the viability of cultures treated with H₂O₂ and PhotoCORM at 14 h to each of the other conditions, *p < 0.05. H₂O₂, hydrogen peroxide.

FIG. 8.

Toxicity from the combination of activated PhotoCORM and H₂O₂ is alleviated by thiourea in cultures of EC958. Cultures were grown in Fe-depleted glucose minimal medium at 37°C, 200 rpm. (A): 0 (●), 50 (■), 100 (▲), 200 (▼), 300 (♦), and 500 () μM PhotoCORMs. In (B), PhotoCORM was added as in (A), followed by being exposed to UV light (365 nm) for 6 min. (C) is as (B), but thiourea (80 mM) was added to cultures before the treatment with PhotoCORM. In (D), 0 (●), 6 (■), 7 (▲), 8 (▼), 9 (♦), and 10 () mM H₂O₂ was added. In (E), cultures treated with PhotoCORM (100 μM) 4 (□), 5 (▲), 6 (▼), 7 (♦), and 8 () mM H₂O₂ were exposed to UV for 6 min and compared with an untreated control (●). (F) is as (E), but thiourea (80 mM) was added to all cultures before the addition of PhotoCORM and H₂O₂. (G) is as (A), but CO-depleted PhotoCORM was added instead. In (H), cultures treated with CO-depleted PhotoCORM (100 μM) were supplemented with 4 (■), 5 (▲), 6 (▼), 7 (♦), and 8 () mM H₂O₂ and compared with an untreated control (●). (I) is as (H), but thiourea (80 mM) was added to all cultures before the addition of CO-depleted PhotoCORM and H₂O₂. Compounds were added at time zero (arrows). Bars represent the standard error of at least three independent experiments.

FIG. 9.

The combination of PhotoCORM and H₂O₂ produces hydroxyl radicals. Fluorescence was measured in glucose minimal medium (A–C) or Fe-depleted glucose minimal medium (D–F). Samples containing PhotoCORMs were exposed to UV light for 6 min (PhotoCORM, UV) or kept in the dark (PhotoCORM dark). PhotoCORM and CO-depleted PhotoCORM final concentrations were 10 μM. HPF (5 μM) was added after the activation of the PhotoCORM, the addition of PhotoCORM dark or CO-depleted PhotoCORM, and before the addition of H₂O₂ (300 μM). EDTA or thiourea (5 and 3 mM final concentration, respectively) was added to samples before the addition of the PhotoCORM or CO-depleted PhotoCORM. EDTA, ethylenediaminetetraacetic acid; HPF, 3′-(p-hydroxyphenyl) fluorescein.

FIG. 10.

Schematic visualization of the activities of PhotoCORM against E. coli strain EC958. Light activation of the PhotoCORM at 365 nm leads to release of the CO ligands from the manganese coordination sphere. The resulting Mn complex is not transported inward , while CO enters the cell via passive diffusion , and inhibits NADH-supported respiration by competing with oxygen, thereby restricting ATP generation. ROS may be formed. CO binds to cytoplasmic heme proteins (not shown) and is sensed by TFs , resulting in transcriptional changes in, for example, genes involved in metal acquisition . Following the loss of CO, the compound reacts with hydrogen peroxide, exogenous, or metabolism derived , forming cytotoxic products such as hydroxyl radicals that perturb membrane integrity. The symbol L indicates the diverse solvent- or biomolecule-derived ligands that take the position of the released carbon monoxide. ROS, reactive oxygen species; TF, transcription factor.