Transition metal carbonyl complexes used as CO-releasing molecules (CORMs) for biological and therapeutic applications may exhibit interesting antimicrobial activity. However, understanding the ...chemical traits and mechanisms of action that rule this activity is required to establish a rationale for the development of CORMs into useful antibiotics. In this work the bactericidal activity, the toxicity to eukaryotic cells, and the ability of CORMs to deliver CO to bacterial and eukaryotic cells were analysed for a set of seven CORMs that differ in the transition metal, ancillary ligands and the CO release profile. Most of these CORMs exhibited bactericidal properties that decrease in the following order: CORM-2 > CORM-3 > ALF062 > ALF850 > ALF186 > ALF153 > Fe(SBPy3)(CO)(BF4)2. A similar yet not entirely coincident decreasing order was found for their induction of intracellular reactive oxygen species (ROS) in E. coli. In contrast, studies in model animal cells showed that for any given CORM, the level of intracellular ROS generated was negligible when compared with that measured inside bacteria. Importantly, these CORMs were in general not toxic to eukaryotic cells, namely murine macrophages, kidney LLC-PK1 epithelial cells, and liver cell line HepG2. CORM-2 and CORM-3 delivered CO to the intracellular space of both E. coli and the two types of tested eukaryotic cells, yet toxicity was only elicited in the case of E. coli. CO delivered by ALF186 into the intercellular space did not enter E. coli cells and the compound was not toxic to either bacteria or to eukaryotic cells. The Fe(ii) carbonyl complex Fe(SBPy3)(CO)(2+) had the reverse, undesirable toxicity profile, being unexpectedly toxic to eukaryotic cells and non-toxic to E. coli. ALF153, the most stable complex in the whole set, was essentially devoid of toxicity or ROS induction ability in all cells. These results suggest that CORMs have a relevant therapeutic potential as antimicrobial drugs since (i) they can show opposite toxicity profiles towards bacteria and eukaryotic cells; (ii) their activity can be modulated through manipulation of the ancillary ligands, as shown with the three {Ru(CO)3}(2+) and two zerovalent Mo based CORMs; and (iii) their toxicity to eukaryotic cells can be made acceptably low. With this new approach, this work contributes to the understanding of the roots of the bactericidal action of CORMs and helps in establishing strategies for their development into a new class of antibiotics.
Helicobacter pylori is a pathogen that establishes long life infections responsible for chronic gastric ulcer diseases and a proved risk factor for gastric carcinoma. The therapeutic properties of ...carbon-monoxide releasing molecules (CORMs) led us to investigate their effect on H. pylori. We show that H. pylori 26695 is susceptible to two widely used CORMs, namely CORM-2 and CORM-3. Also, several H. pylori clinical isolates were killed by CORM-2, including those resistant to metronidazole. Moreover, sub-lethal doses of CORM-2 combined with metronidazole, amoxicillin and clarithromycin was found to potentiate the effect of the antibiotics. We further demonstrate that the mechanisms underpinning the antimicrobial effect of CORMs involve the inhibition of H. pylori respiration and urease activity. In vivo studies done in key cells of the innate immune system, such as macrophages, showed that CORM-2, either alone or when combined with metronidazole, strongly reduces the ability of H. pylori to infect animal cells. Hence, CORMs have the potential to kill antibiotic resistant strains of H. pylori.
CO-releasing molecules (CO-RMs) were previously shown by us to be more potent bactericides than CO gas. This suggests a mechanism of action for CO-RM, which either potentiates the activity of CO or ...uses another CO-RM-specific effect. We have also reported that CORM-2 induces the expression of genes related to oxidative stress. In the present study we intend to establish whether the generation of reactive oxygen species by CO-RMs may indeed result in the inhibition of bacterial cellular function. We now report that two CO-RMs (CORM-2 and ALF062) stimulate the production of ROS in Escherichia coli, an effect that is abolished by addition of antioxidants. Furthermore, deletion of genes encoding E. coli systems involved in reactive oxygen species scavenging, namely catalases and superoxide dismutases, potentiates the lethality of CORM-2 due to an increase of intracellular ROS content. CORM-2 also induces the expression of the E. coli DNA repair/SOS system recA, and its inactivation enhances toxicity of CORM-2. Moreover, fluorescence microscopy images reveal that CORM-2 causes DNA lesions to bacterial cells. We also demonstrate that cells treated with CORM-2 contain higher levels of free iron arising from destruction of iron-sulfur proteins. Importantly, we show that CO-RMs generate hydroxyl radicals in a cell-free solution, a process that is abolished by scavenging CO. Altogether, we provide a novel insight into the molecular basis of CO-RMs action by showing that their bactericidal properties are linked to cell damage inflicted by the oxidative stress that they are able to generate.
Escherichia coli RIC (Repair of Iron Centers) is a diiron protein previously reported to be involved in the repair of iron-sulfur proteins damaged by oxidative or nitrosative stresses, and proposed ...to act as an iron donor. This possible role of RIC was now examined specifically by evaluating its ability to donate iron ions to apo-iron-sulfur proteins, determining the iron binding constants and assessing the lability of its iron ions. We show, by UV-visible, EPR and resonance Raman spectroscopies that RIC may participate in the synthesis of an iron-sulfur cluster in the apo-forms of the spinach ferredoxin and IscU when in the presence of the sulfide donating system IscS and L-cysteine. Iron binding assays allowed determining the as-isolated and fully reduced RIC dissociation constants for the ferric and ferrous iron of 10-27 M and 10-13 M, respectively. Mössbauer studies revealed that the RIC iron ions are labile, namely when the center is in the mixed-valence redox form as compared with the (μ-oxo) diferric one. Altogether, these results suggest that RIC is capable of delivering iron for the formation of iron-sulfur clusters.
Staphylococcus aureus
is a pathogen responsible for severe community- and nosocomially acquired infections. To fight pathogen intrusion, the innate immune system uses a plethora of weapons, with the ...generation of oxidative and nitrosative stresses among the most efficient. In this work, the
S. aureus
genome-wide transcriptional responses to oxidative stress generated by hydrogen peroxide, to nitrosative stress imposed by
S
-nitrosoglutathione (GSNO), and to the combination of the two were investigated using microarray analysis. The results showed that these stresses have a significant impact on the transcriptome of
S. aureus
. Hydrogen peroxide modified mainly the mRNA abundance of genes involved in oxidative detoxification and DNA metabolism, which together represent 14 % of the total number of upregulated genes. GSNO caused significant alteration of the expression of gene products with regulatory function. However, the simultaneous addition of GSNO and hydrogen peroxide was found to cause the more significant transcriptomic alteration, affecting ∼10 % of the total transcriptome. In particular, exposure of
S. aureus
to GSNO plus hydrogen peroxide modified the transcription of genes associated with cell envelope and iron metabolism, including induction of
ftnA
and
dps
genes that encode iron-storage and oxidative-protecting proteins. Further studies revealed that when exposed to combined GSNO–hydrogen peroxide stresses,
S. aureus
has decreased viability, which is enhanced in the presence of iron, and low siderophore activity. Altogether, this study revealed, for the first time, how the combined oxidative and nitrosative stresses inflicted during phagocytosis interfere at the transcriptional level with the
S. aureus
cellular metabolism.
Abstract
Carbon monoxide-releasing molecules (CO-RMs) are, in general, transition metal carbonyl complexes that liberate controlled amounts of CO. In animal models, CO-RMs have been shown to reduce ...myocardial ischaemia, inflammation and vascular dysfunction, and to provide a protective effect in organ transplantation. Moreover, CO-RMs are bactericides that kill both Gram-positive and Gram-negative bacteria such as Staphylococcus aureus and Pseudomonas aeruginosa. Herein are reviewed the microbial genetic and biochemical responses associated with CO-RM-mediated cell death. Particular emphasis is given to the data revealing that CO-RMs induce the generation of reactive oxygen species (ROS), which contribute to the antibacterial activity of these compounds.
1 Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, Avenida da República (EAN), 2780-157 Oeiras, Portugal
2 Department of Bioinformatics, Centro de Investigación Príncipe ...Felipe (CIPF), Valencia, E-46013, Spain
3 Center for Biomedical Research on Rare Diseases (CIBERER), Centro de Investigación Príncipe Felipe (CIPF), Valencia, E-46013, Spain
4 Functional Genomics Node (National Institute for Bioinformatics, INB), Centro de Investigación Príncipe Felipe (CIPF), Valencia, E-46013, Spain
Correspondence Lígia M. Saraiva lst{at}itqb.unl.pt
We recently reported that carbon monoxide (CO) has bactericidal activity. To understand its mode of action we analysed the gene expression changes occurring when Escherichia coli , grown aerobically and anaerobically, is treated with the CO-releasing molecule CORM-2 (tricarbonyldichlororuthenium(II) dimer). Microarray analysis shows that the E. coli CORM-2 response is multifaceted, with a high number of differentially regulated genes spread through several functional categories, namely genes involved in inorganic ion transport and metabolism, regulators, and genes implicated in post-translational modification, such as chaperones. CORM-2 has a higher impact in E. coli cells grown anaerobically, as judged by the repression of genes belonging to eight functional classes which are not seen in the response of aerobically CORM-2-treated cells. The biological relevance of the variations caused by CORM-2 was substantiated by studying the CORM-2 sensitivity of selected E. coli mutants. The results show that the deletion of redox-sensing regulators SoxS and OxyR increased the sensitivity to CORM-2 and suggest that while SoxS plays an important role in protection against CORM-2 under both growth conditions, OxyR seems to participate only in the aerobic CORM-2 response. Under anaerobic conditions, we found that the heat-shock proteins IbpA and IbpB contribute to CORM-2 defence since the deletion of these genes increases the sensitivity of the strain. The induction of several met genes and the hypersensitivity to CORM-2 of the metR , metI and metN mutant strains suggest that CO has effects on the methionine metabolism of E. coli . CORM-2 also affects the transcription of several E. coli biofilm-related genes and increases biofilm formation in E. coli . In particular, the absence of tqsA or bhsA increases the resistance of E. coli to CORM-2, and deletion of tsqA leads to a strain that has lost its capacity to form biofilm upon treatment with CORM-2. In spite of the relatively stable nature of the CO molecule, our results show that CO is able to trigger a significant alteration in the transcriptome of E. coli which necessarily has effects in several key metabolic pathways.
Abbreviations: CORM, CO-releasing molecule; CORM-2, tricarbonyldichlororuthenium(II) dimer; FDR, false discovery rate
The GEO accession number for the microarray data associated with this paper is GSE13982.
Six supplementary tables are available with the online version of this paper.
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