Marta C. Justino

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.

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.

Iron–sulfur repair YtfE protein from Escherichia coli : structural characterization of the di-iron center

YtfE was recently shown to be a newly discovered protein required for the recovery of the activity of iron–sulfur-containing enzymes damaged by oxidative and nitrosative stress conditions. The Escherichia coli YtfE purified protein is a dimer with two iron atoms per monomer and the type and properties of the iron center were investigated by using a combination of resonance Raman and extended X-ray absorption fine structure spectroscopies. The results demonstrate that YtfE contains a non-heme dinuclear iron center having μ-oxo and μ-carboxylate bridging ligands and six histidine residues coordinating the iron ions. This is the first example of a protein from this important class of di-iron proteins to be shown to be involved in the repair of iron–sulfur centers.

Di-iron proteins of the Ric family are involved in iron–sulfur cluster repair

A key element in eukaryotic immune defenses against invading microbes is the production of reactive oxygen and nitrogen species. One of the main targets of these species are the iron–sulfur clusters, which are essential prosthetic groups that confer to proteins the ability to perform crucial roles in biological processes. Microbes have developed sophisticated systems to eliminate nitrosative and oxidative species and promote the repair of the damages inflicted. The Ric (Repair of Iron Centers) proteins constitute a novel family of microbial di-iron proteins with a widespread distribution among microbes, including Gram-positive and Gram-negative bacteria, protozoa and fungi. The Ric proteins are encoded by genes that are up-regulated by nitric oxide and hydrogen peroxide. Recent studies have shown that the active di-iron center is involved in the restoration of Fe–S clusters damaged by exposure to nitric oxide and hydrogen peroxide.

Detection by whole genome microarrays of a spontaneous 126-gene deletion during construction of a ytfE mutant: confirmation that a ytfE mutation results in loss of repair of iron-sulfur centres in proteins damaged by oxidative or nitrosative stress.

We show that genomic hybridization allows detection of a spontaneous secondary deletion of 126 genes that occurred during construction of an Escherichia coli ytfE mutant, LMS4209, explaining some of its unexpected growth defects. We confirm that YtfE is required to repair damage to iron-sulfur centres and for hydrogen peroxide resistance.

Helicobacter pylori Has an Unprecedented Nitric Oxide Detoxifying System

AIMS The ability of pathogens to cope with the damaging effects of nitric oxide (NO), present in certain host niches and produced by phagocytes that support innate immunity, relies on multiple strategies that include the action of detoxifying enzymes. As for many other pathogens, these systems remained unknown for Helicobacter pylori. This work aimed at identifying and functionally characterizing an H. pylori system involved in NO protection. RESULTS In the present work, the hp0013 gene of H. pylori is shown to be related to NO resistance, as its inactivation increases the susceptibility of H. pylori to nitrosative stress, and significantly decreases the NADPH-dependent NO reduction activity of H. pylori cells. The recombinant HP0013 protein is able to complement an NO reductase-deficient Escherichia coli strain and exhibits significant NO reductase activity. Mutation of hp0013 renders H. pylori more vulnerable to nitric oxide synthase-dependent macrophage killing, and decreases the ability of the pathogen to colonize mice stomachs. INNOVATION Phylogenetic studies reveal that HP0013, which shares no significant amino acid sequence similarity to the other so far known microbial NO detoxifiers, belongs to a novel family of proteins with a widespread distribution in the microbial world. CONCLUSION H. pylori HP0013 represents an unprecedented enzymatic NO detoxifying system for the in vivo microbial protection against nitrosative stress.

Oxidative Stress Modulates the Nitric Oxide Defense Promoted by Escherichia coli Flavorubredoxin

Mammalian cells of innate immunity respond to pathogen invasion by activating proteins that generate a burst of oxidative and nitrosative stress. Pathogens defend themselves from the toxic compounds by triggering a variety of detoxifying enzymes. Escherichia coli flavorubredoxin is a nitric oxide reductase that is expressed under nitrosative stress conditions. We report that in contrast to nitrosative stress alone, exposure to both nitrosative and oxidative stresses abolishes the expression of flavorubredoxin. Electron paramagnetic resonance (EPR) experiments showed that under these conditions, the iron center of the flavorubredoxin transcription activator NorR loses the ability to bind nitric oxide. Accordingly, triggering of the NorR ATPase activity, a requisite for flavorubredoxin activation, was impaired by treatment of the protein with the double stress. Studies of macrophages revealed that the contribution of flavorubredoxin to the survival of E. coli depends on the stage of macrophage infection and that the lack of protection observed at the early phase is related to inhibition of NorR activity by the oxidative burst. We propose that the time-dependent activation of flavorubredoxin contributes to the adaptation of E. coli to the different fluxes of hydrogen peroxide and nitric oxide to which the bacterium is subjected during the course of macrophage infection.

The Bactericidal Activity of Carbon Monoxide-Releasing Molecules against Helicobacter pylori

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.

FrxA is an S-nitrosoglutathione reductase enzyme that contributes to Helicobacter pylori pathogenicity

Helicobacter pylori is a pathogen that infects the gastric mucosa of a large percentage of the human population worldwide, and predisposes to peptic ulceration and gastric cancer. Persistent colonization of humans by H. pylori triggers an inflammatory response that leads to the production of reactive nitrogen species. However, the mechanisms of H. pylori defence against nitrosative stress remain largely unknown. In this study, we show that the NADH‐flavin oxidoreductase FrxA of H. pylori, besides metabolizing nitrofurans and metronidazole, has S‐nitrosoglutathione reductase activity. In agreement with this, inactivation of the FrxA‐encoding gene resulted in a strain that was more sensitive to S‐nitrosoglutathione. FrxA was also shown to contribute to the proliferation of H. pylori in macrophages, which are key phagocytic cells of the mammalian innate immune system. Moreover, FrxA was shown to support the virulence of the pathogen upon mouse infection. Altogether, we provide evidence for a new function of FrxA that contributes to the successful chronic colonization ability that characterizes H. pylori.

Camphor-based CCR5 blocker lead compounds – a computational and experimental approach

The C–C chemokine receptor type 5 (CCR5) is a transmembrane receptor that plays a pivotal role as a HIV anchor to human cell membranes, mediating viral entry. CCR5 antagonists, acting by blocking the receptor and preventing its interaction with the HIV proteins, are key agents towards effective anti-viral therapy. This work describes the computational study, synthesis and viral inhibition assay of a number of camphor derivatives as a first step towards new drug leads to block this specific entry pathway. Viral inhibition assays have identified three molecules, camphor carboxylic acid, its tri(hydroxymethyl)aminomethane amide derivative, and an hydroxyl-imide camphor derivative as promising agents to develop new drugs, with IC50 values (0.16, 0.22 and 1.02 μM, respectively) one order below that of maraviroc (0.02 μM), a clinically used CCR5 antagonist.