Marta Justino

<p>(<b>A</b>) MIC of metronidazole determined for each <i>H pylori</i> clinical isolate in the absence (grey) and in the presence of CORM-2 (black). Strains 5846 and 5587 were treated 50 mg/CORM-2, strains 5611 and 4574 exposed to 100 mg/L CORM-2, and strains 5599 and 4597 submitted to 150 mg/L CORM-2. (<b>B</b>) MBC of metronidazole for each <i>H pylori</i> clinical isolates, in the absence (grey) and in the presence of CORM-2 (black). Strains 5846 and 5587 were treated 200 mg/CORM-2, and strains 5611, 4574, 5599 and 4597 exposed to 150 mg/L CORM-2. For each strain, metronidazole was combined with a CORM-2 concentration below the MIC/MBC of the CORM-2 alone. In all cases, values representing the median of five biological samples were significantly different (p<0.05 in Mann Whitney <i>t</i> test).</p

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 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.

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 mu-oxo and mu-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.

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.

The flavodiiron proteins (FDPs), present in Archaea, Bacteria, and some protozoan pathogens (mostly anaerobes or microaerophiles), have been proposed to afford protection to microbes against nitric oxide and/or oxygen (toxic for anaerobes). The structural prototype of this protein family is a homodimer assembled in a "head-to-tail" configuration, with each monomer being composed of two domains: an N-terminal metallo-beta-lactamase module harboring a nonheme diiron center (active site of NO/O(2) reduction) and a C-terminal flavodoxin module, where a flavin mononucleotide moiety is embedded. Several FDPs bear C-terminal extra domains, which influence the composition of the respective electron transfer chains that couple NAD(P)H oxidation to NO/O(2) reduction. Herein are described methodologies employed to successfully produce, isolate, and characterize fully operative recombinant flavodiiron proteins. Spectroscopic techniques, namely absorption (visible and near-ultraviolet) and electron paramagnetic resonance spectroscopies, allowed redox-sensitive spectral fingerprints to be obtained, used further in the functional characterization of isolated flavodiiron proteins. Altogether, these studies on pure proteins contribute to understanding the molecular determinants that govern the in vivo function of the FDPs.

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. 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. 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. H. pylori HP0013 represents an unprecedented enzymatic NO detoxifying system for the in vivo microbial protection against nitrosative stress.