Lígia M. Saraiva

Antimicrobial Action of Carbon Monoxide-Releasing Compounds

ABSTRACT Carbon monoxide (CO) is endogenously produced in the human body, mainly from the oxidation of heme catalyzed by heme oxygenase (HO) enzymes. The induction of HO and the consequent increase in CO production play important physiological roles in vasorelaxation and neurotransmission and in the immune system. The exogenous administration of CO gas and CO-releasing molecules (CO-RMs) has been shown to induce vascular effects and to alleviate hypoxia-reoxygenation injury of mammalian cells. In particular, due to its anti-inflammatory, antiapoptotic, and antiproliferative properties, CO inhibits ischemic-reperfusion injury and provides potent cytoprotective effects during organ and cell transplantation. In spite of these findings regarding the physiology and biology of mammals, nothing is known about the action of CO on bacteria. In the present work, we examined the effect of CO on bacterial cell proliferation. Cell growth experiments showed that CO caused the rapid death of the two pathogenic bacteria tested, Escherichia coli and Staphylococcus aureus, particularly when delivered through organometallic CO-RMs. Of importance is the observation that the effectiveness of the CO-RMs was greater in near-anaerobic environments, as many pathogens are anaerobic organisms and pathogen colonization occurs in environments with low oxygen concentrations. Our results constitute the first evidence that CO can be utilized as an antimicrobial agent. We anticipate our results to be the starting point for the development of novel types of therapeutic drugs designed to combat antibiotic-resistant pathogens, which are widespread and presently a major public health concern.

Escherichia coli Di-iron YtfE Protein Is Necessary for the Repair of Stress-damaged Iron-Sulfur Clusters

DNA microarray experiments showed that the expression of the Escherichia coli ytfE gene is highly increased upon exposure to nitric oxide. We also reported that deletion of ytfE significantly alters the phenotype of E. coli, generating a strain with enhanced susceptibility to nitrosative stress and defective in the activity of several iron-sulfur-containing proteins. In this work, it is shown that the E. coli ytfE confers protection against oxidative stress. Furthermore, we found that the damage of the [4Fe-4S]2+ clusters of aconitase B and fumarase A caused by exposure to hydrogen peroxide and nitric oxide stress occurs at higher rates in the absence of ytfE. The ytfE null mutation also abolished the recovery of aconitase and fumarase activities, which is observed in wild type E. coli once the stress is scavenged. Notably, upon the addition of purified holo-YtfE protein to the mutant cell extracts, the enzymatic activities of fumarase and aconitase are fully recovered and at rates similar to the wild type strain. We concluded that YtfE is critical for the repair of iron-sulfur clusters damaged by oxidative and nitrosative stress conditions.

A novel membrane-bound respiratory complex from Desulfovibrio desulfuricans ATCC 27774.

In the anaerobic respiration of sulfate, performed by sulfate-reducing prokaryotes, reduction of the terminal electron acceptor takes place in the cytoplasm. The membrane-associated electron transport chain that feeds electrons to the cytoplasmic reductases is still very poorly characterized. In this study we report the isolation and characterization of a novel membrane-bound redox complex from Desulfovibrio desulfuricans ATCC 27774. This complex is formed by three subunits, and contains two hemes b, two FAD groups and several iron-sulfur centers. The two hemes b are low-spin, with macroscopic redox potentials of +75 and -20 mV at pH 7.6. Both hemes are reduced by menadiol, a menaquinone analogue, indicating a function for this complex in the respiratory electron-transport chain. EPR studies of the as-isolated and dithionite-reduced complex support the presence of a [3Fe-4S](1+/0) center and at least four [4Fe-4S](2+/1+) centers. Cloning of the genes coding for the complex subunits revealed that they form a putative transcription unit and have homology to subunits of heterodisulfide reductases (Hdr). The first and second genes code for soluble proteins that have homology to HdrA, whereas the third gene codes for a novel type of membrane-associated protein that contains both a hydrophobic domain with homology to the heme b protein HdrE and a hydrophilic domain with homology to the iron-sulfur protein HdrC. Homologous operons are found in the genomes of other sulfate-reducing organisms and in the genome of the green-sulfur bacterium Chlorobium tepidum TLS. The isolated complex is the first example of a new family of respiratory complexes present in anaerobic prokaryotes.

Entamoeba histolytica modulates a complex repertoire of novel genes in response to oxidative and nitrosative stresses: implications for amebic pathogenesis.

Upon host infection, the protozoan parasite Entamoeba histolytica is confronted with reactive oxygen and nitrogen species and must survive these stresses in order to cause invasive disease. We analysed the parasites response to oxidative and nitrosative stresses, probing the transcriptional changes of trophozoites of a pathogenic strain after a 60 min exposure to H2O2 (1 mM) or a NO donor (dipropylenetriamine‐NONOate, 200 μM), using whole‐genome DNA microarrays. Genes encoding reactive oxygen and nitrogen species detoxification enzymes had high transcriptional levels under basal conditions and upon exposure to both stresses. On a whole‐genome level, there was significant modulation of gene expression by H2O2 (286 genes regulated) and dipropylenetriamine‐NONOate (1036 genes regulated) with a significant overlap of genes modulated under both conditions (164 genes). A number of transcriptionally regulated genes were in signalling/regulatory and repair/metabolic pathways. However, the majority of genes with altered transcription encode unknown proteins, suggesting as yet unraveled response pathways in E. histolytica. Trophozoites of a non‐pathogenic E. histolytica strain had a significantly muted transcriptional response to H2O2 compared with the pathogenic strain, hinting that differential response to oxidative stress may be one factor that contributes to the pathogenic potential of E. histolytica.

Reactive Oxygen Species Mediate Bactericidal Killing Elicited by Carbon Monoxide-releasing Molecules

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.

The role of the flavodiiron proteins in microbial nitric oxide detoxification

The flavodiiron proteins (first named as A-type flavoproteins) constitute a large superfamily of enzymes, widespread among anaerobic and facultative anaerobic prokaryotes, from both the Archaea and Bacteria domains. Noticeably, genes encoding for homologous enzymes are also present in the genomes of some pathogenic and anaerobic amitochondriate protozoa. The fingerprint of this enzyme family is the conservation of a two-domain structural core, built by a metallo-beta-lactamase-like domain, at the N-terminal region, harbouring a non-heme diiron site, and a flavodoxin-like domain, containing one FMN moiety. These enzymes have a significant nitric oxide reductase activity, and there is increasing evidence that they are involved in microbial resistance to nitric oxide. In this review, we will discuss available data for this novel family of enzymes, including their physicochemical properties, structural and phylogenetic analyses, enzymatic properties and the molecular genetic approaches so far used to tackle their function.

The Role of the Hybrid Cluster Protein in Oxidative Stress Defense

Hybrid cluster proteins (HCP) contain two types of Fe/S clusters, namely a [4Fe-4S]2+/1+ or [2Fe-2S]2+/1+ cluster and a novel type of hybrid cluster, [4Fe-2S-2O], in the as-isolated state. Although first isolated from anaerobic sulfate-reducing bacteria, the analysis of the genomic sequences reveals that genes encoding putative hybrid cluster proteins are present in a wide range of organisms, aerobic, anaerobic, or facultative, from the Bacteria, Archaea, and Eukarya domains. Despite a detailed spectroscopic and structural characterization, the precise physiological function of these proteins remained unknown. The present work shows that the transcription of the Escherichia coli hcp gene is induced by hydrogen peroxide, and this induction is regulated by the redox-sensitive transcriptional activator, OxyR. The E. coli hcp mutant strain exhibits higher sensitivity to hydrogen peroxide, a behavior that reverts to the wild type phenotype once a plasmid carrying the hcp gene is reintroduced. Furthermore, the purified HCPs from E. coli and Desulfovibrio desulfuricans ATCC 27774 show an alternative enzymatic activity, which under physiological conditions exhibited Km values for hydrogen peroxide (∼0.3 mm) within the range of other peroxidases. Altogether, the results reveal that HCP is involved in oxidative stress protection.

Widespread distribution in pathogenic bacteria of di-iron proteins that repair oxidative and nitrosative damage to iron-sulfur centers.

Expression of two genes of unknown function, Staphylococcus aureus scdA and Neisseria gonorrhoeae dnrN, is induced by exposure to oxidative or nitrosative stress. We show that DnrN and ScdA are di-iron proteins that protect their hosts from damage caused by exposure to nitric oxide and to hydrogen peroxide. Loss of FNR-dependent activation of aniA expression and NsrR-dependent repression of norB and dnrN expression on exposure to NO was restored in the gonococcal parent strain but not in a dnrN mutant, suggesting that DnrN is necessary for the repair of NO damage to the gonococcal transcription factors, FNR and NsrR. Restoration of aconitase activity destroyed by exposure of S. aureus to NO or H2O2 required a functional scdA gene. Electron paramagnetic resonance spectra of recombinant ScdA purified from Escherichia coli confirmed the presence of a di-iron center. The recombinant scdA plasmid, but not recombinant plasmids encoding the complete Escherichia coli sufABCDSE or iscRSUAhscBAfdx operons, complemented repair defects of an E. coli ytfE mutant. Analysis of the protein sequence database revealed the importance of the two proteins based on the widespread distribution of highly conserved homologues in both gram-positive and gram-negative bacteria that are human pathogens. We provide in vivo and in vitro evidence that Fe-S clusters damaged by exposure to NO and H2O2 can be repaired by this new protein family, for which we propose the name repair of iron centers, or RIC, proteins.

Molecular hijacking of siroheme for the synthesis of heme and d1 heme.

Modified tetrapyrroles such as chlorophyll, heme, siroheme, vitamin B12, coenzyme F430, and heme d1 underpin a wide range of essential biological functions in all domains of life, and it is therefore surprising that the syntheses of many of these life pigments remain poorly understood. It is known that the construction of the central molecular framework of modified tetrapyrroles is mediated via a common, core pathway. Herein a further branch of the modified tetrapyrrole biosynthesis pathway is described in denitrifying and sulfate-reducing bacteria as well as the Archaea. This process entails the hijacking of siroheme, the prosthetic group of sulfite and nitrite reductase, and its processing into heme and d1 heme. The initial step in these transformations involves the decarboxylation of siroheme to give didecarboxysiroheme. For d1 heme synthesis this intermediate has to undergo the replacement of two propionate side chains with oxygen functionalities and the introduction of a double bond into a further peripheral side chain. For heme synthesis didecarboxysiroheme is converted into Fe-coproporphyrin by oxidative loss of two acetic acid side chains. Fe-coproporphyrin is then transformed into heme by the oxidative decarboxylation of two propionate side chains. The mechanisms of these reactions are discussed and the evolutionary significance of another role for siroheme is examined.

Spontaneous CO Release from RuII(CO)2–Protein Complexes in Aqueous Solution, Cells, and Mice†

We demonstrate that RuII(CO)2–protein complexes, formed by the reaction of the hydrolytic decomposition products of [fac-RuCl(κ2-H2NCH2CO2)(CO)3] (CORM-3) with histidine residues exposed on the surface of proteins, spontaneously release CO in aqueous solution, cells, and mice. CO release was detected by mass spectrometry (MS) and confocal microscopy using a CO-responsive turn-on fluorescent probe. These findings support our hypothesis that plasma proteins act as CO carriers after in vivo administration of CORM-3. CO released from a synthetic bovine serum albumin (BSA)–RuII(CO)2 complex leads to downregulation of the cytokines interleukin (IL)-6, IL-10, and tumor necrosis factor (TNF)-α in cancer cells. Finally, administration of BSA–RuII(CO)2 in mice bearing a colon carcinoma tumor results in enhanced CO accumulation at the tumor. Our data suggest the use of RuII(CO)2–protein complexes as viable alternatives for the safe and spatially controlled delivery of therapeutic CO in vivo.