Carbon Monoxide-releasing Antibacterial Molecules Target Respiration and Global Transcriptional Regulators

Abstract

Carbon monoxide, a classical respiratory inhibitor, also exerts vasodilatory, anti-inflammatory, and antiapoptotic effects. COreleasing molecules have therapeutic value, increasing phagocytosis and reducing sepsis-induced lethality. Here we identify for the first time the bacterial targets of Ru(CO) 3 Cl(glycinate) (CORM-3), a ruthenium-based carbonyl that liberates CO rapidly under physiological conditions. Contrary to the expectation that CO would be preferentially inhibitory at low oxygen tensions or anaerobically, Escherichia coli cultures were also sensitive to CORM-3 at concentrations equimolar with oxygen. CORM-3, assayed as ruthenium, was taken up by bacteria and rapidly delivered CO intracellularly to terminal oxidases. Microarray analysis of CORM-3-treated cells revealed extensively modified gene expression, notably down-regulation of genes encoding key aerobic respiratory complexes. Genes involved in metal metabolism, homeostasis, or transport were also differentially expressed, and free intracellular zinc levels were elevated. Probabilistic modeling of transcriptomic data identified the global transcription regulators ArcA, CRP, Fis, FNR, Fur, BaeR, CpxR, and IHF as targets and potential CO sensors. Our discovery that CORM-3 is an effective inhibitor and global regulator of gene expression, especially under aerobic conditions, has important implications for administration of CO-releasing agents in sepsis and inflammation.It has been recognized for over 80 years that carbon monoxide (CO) combines with ferrous hemoglobin, competing with oxygen and inhibiting respiration (1). Recently, however, CO has been shown to have unexpected and profound physiological effects in higher organisms. Inducible heme oxygenase-1 and constitutive heme oxygenase-2 degrade heme to generate CO that has vasodilatory, antiflammatory, and antiapoptotic effects (2). The CO is thought to target transition elements in biological systems, particularly hemes (in oxidases, globins, and CO sensors, for example) (3), nickel (in CO dehydrogenase), and complex nickel-, iron-and sulfur-containing clusters (2). The impact of CO in physiology and medicine has prompted development of metal carbonyl compounds (carbon monoxide-releasing molecules; CO-RMs) 2 to deliver CO in biological environments. Such molecules are pharmacologically active, eliciting vasodilation in isolated aorta and mediating hypotension in vivo (4). Newer water-soluble CO-RMs have revealed roles for CO in suppression of inflammation, protection against hypoxia-reoxygenation and oxidative stress, and mitigation of myocardial infarction and other disorders (5).Considering the potent biological activities of CO and CORMs, we know little about CO toxicity toward microorganisms, despite CO being a stable gas that might find application in antimicrobial therapies. Indeed, recent studies in inflammatory models of disease, such as endotoxin exposure, suggest that heme oxygenase-1 and its products exert beneficial effects in mice (6 -8). Although biliverdin (9) generated by HO ac...

Compensations for Diminished Terminal Oxidase Activity in Escherichia coli

“…By using a variational approximation, the inference problem can be approximately solved, providing estimates of the changes in activity of regulators (with error bars) from an analysis of the behavior of their targets. This technique has been applied to reconstructing the regulatory response in E. coli to the transition between aerobic and microaerobic conditions (33) and to the response to CO-releasing molecules (34). The statistical significance of the results was assessed by running the model 100 times on the same data but using a randomly generated regulatory network (with identical average connectivity), as described previously (34).…”

“…Following the same procedure used in Ref. 34, we estimated the probability of obtaining as many as six significantly changing regulators by repeating the analysis with 100 different randomizations of the regulatory network. By fitting a geometric distribution to the resulting histogram, we could estimate that the probability of obtaining six or more regulators significantly changing in a random control is smaller than 0.001.…”

Live-Fibroblast IR Imaging of a Cytoprotective PhotoCORM Activated with Visible Light

“…[18][19][20][21] While solutions of CO gas also impair bacterial growth, they do not match the effectiveness of the CO releasing molecule. In this case the pronounced bactericidal effects of CORM-3 are ascribed to the ability of the molecule to specifically deliver intracellular CO to heme-containing oxidase(s) and to facilitate the electrogenic transmembrane movement of K+ (or Na+) ions.…”

Analysis of the Bacterial Response to Ru(CO)3Cl(Glycinate) (CORM-3) and the Inactivated Compound Identifies the Role Played by the Ruthenium Compound and Reveals Sulfur-Containing Species as a Major Target of CORM-3 Action

“…CORM-3 is bactericidal against a variety of microbes, including Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus (10,12,13,30,40).…”