Pseudomonas aeruginosa Bacterioferritin Is Assembled from FtnA and BfrB Subunits with the Relative Proportions Dependent on the Environmental Oxygen Availability

Ferritins are iron storage proteins assembled from 24 subunits into a spherical and hollow structure. The genomes of many bacteria harbor genes encoding two types of ferritin-like proteins, the bacterial ferritins (Ftn) and the bacterioferritins (Bfr), which bind heme. The genome of P. aeruginosa PAO1 (like the genomes of many bacteria) contains genes coding for two different types of ferritin-like molecules, ftnA (PA4235) and bfrB (PA3531). The reasons for requiring the presence of two distinct types of iron storage protein in bacterial cells have remained largely unexplained. Attempts to understand this issue in P. aeruginosa through the recombinant expression of the ftnA and bfrB genes in E. coli host cells, coupled to the biochemical and structural characterization of the recombinant 24-mer FtnA and 24-mer BfrB molecules, have shown that each of the recombinant molecules can form an Fe3+-mineral core. These observations led to the suggestion that 24-mer FtnA and 24-mer BfrB molecules coexist in P. aeruginosa cells where they share iron storage responsibilities. Herein, we demonstrate that P. aeruginosa utilizes a single heterooligomeric 24-mer Bfr assembled from FtnA and BfrB subunits. The relative content of the FtnA and BfrB subunits in Bfr depends on the O2 availability during cell culture, such that Bfr isolated from aerobically cultured P. aeruginosa is assembled from a majority of BfrB subunits. In contrast, when the cells are cultured in O2-limiting conditions, the proportion of FtnA subunits in the isolated Bfr increases significantly and can become the most abundant subunit. Despite the variability in the subunit composition of Bfr, the 24-mer assembly is consistently arranged from FtnA subunit dimers devoid of heme and BfrB subunit dimers each containing a heme molecule

Structural and mutational analyses of the Leptospira interrogans virulence-related heme oxygenase provide insights into its catalytic mechanism

© 2017 Soldano et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Heme oxygenase from Leptospira interrogans is an important virulence factor. During catalysis, redox equivalents are provided to this enzyme by the plastidic-type ferredoxin-NADP+ reductase also found in L. interrogans. This process may have evolved to aid this bacterial pathogen to obtain heme-iron from their host and enable successful colonization. Herein we report the crystal structure of the heme oxygenase-heme complex at 1.73 Å resolution. The structure reveals several distinctive features related to its function. A hydrogen bonded network of structural water molecules that extends from the catalytic site to the protein surface was cleared observed. A depression on the surface appears to be the H+ network entrance from the aqueous environment to the catalytic site for O2 activation, a key step in the heme oxygenase reaction. We have performed a mutational analysis of the F157, located at the above-mentioned depression. The mutant enzymes were unable to carry out the complete degradation of heme to biliverdin since the reaction was arrested at the verdoheme stage. We also observed that the stability of the oxyferrous complex, the efficiency of heme hydroxylation and the subsequent conversion to verdoheme was adversely affected. These findings underscore a long-range communication between the outer fringes of the hydrogen-bonded network of structural waters and the heme active site during catalysis. Finally, by analyzing the crystal structures of ferredoxin-NADP+ reductase and heme oxygenase, we propose a model for the productive association of these proteins

Pseudomonas aeruginosa Dps (PA0962) Functions in H2O2 Mediated Oxidative Stress Defense and Exhibits In Vitro DNA Cleaving Activity

We report the structural, biochemical, and functional characterization of the product of gene PA0962 from Pseudomonas aeruginosa PAO1. The protein, termed Pa Dps, adopts the Dps subunit fold and oligomerizes into a nearly spherical 12-mer quaternary structure at pH 6.0 or in the presence of divalent cations at neutral pH and above. The 12-Mer Pa Dps contains two di-iron centers at the interface of each subunit dimer, coordinated by conserved His, Glu, and Asp residues. In vitro, the di-iron centers catalyze the oxidation of Fe2+ utilizing H2O2 (not O2) as an oxidant, suggesting Pa Dps functions to aid P. aeruginosa to survive H2O2-mediated oxidative stress. In agreement, a P. aeruginosa Δdps mutant is significantly more susceptible to H2O2 than the parent strain. The Pa Dps structure harbors a novel network of Tyr residues at the interface of each subunit dimer between the two di-iron centers, which captures radicals generated during Fe2+ oxidation at the ferroxidase centers and forms di-tyrosine linkages, thus effectively trapping the radicals within the Dps shell. Surprisingly, incubating Pa Dps and DNA revealed unprecedented DNA cleaving activity that is independent of H2O2 or O2 but requires divalent cations and 12-mer Pa Dps

Inhibiting Iron Mobilization from Bacterioferritin in Pseudomonas aeruginosa Impairs Biofilm Formation Irrespective of Environmental Iron Availability

Copyright © 2020 American Chemical Society. Although iron is essential for bacteria, the nutrient presents problems of toxicity and solubility. Bacteria circumvent these problems with the aid of iron storage proteins where Fe3+ is deposited and, when necessary, mobilized as Fe2+ for metabolic requirements. In Pseudomonas aeruginosa, Fe3+ is compartmentalized in bacterioferritin (BfrB), and its mobilization as Fe2+ requires specific binding of a ferredoxin (Bfd) to reduce the stored Fe3+. Blocking the BfrB-Bfd complex leads to irreversible iron accumulation in BfrB and cytosolic iron deprivation. Consequently, given the intracellular iron sufficiency requirement for biofilm development, we hypothesized that blocking the BfrB-Bfd interaction in P. aeruginosa would impair biofilm development. Our results show that planktonic and biofilm-embedded cells where the BfrB-Bfd complex is blocked exhibit cytosolic iron deficiency, and poorly developed biofilms, even in iron-sufficient culture conditions. These results underscore inhibition of the BfrB-Bfd complex as a rational target to dysregulate iron homeostasis and possibly control biofilms

Heme-iron utilization by Leptospira interrogans requires a heme oxygenase and a plastidic-type ferredoxin-NADP+ reductase

Background: Heme oxygenase catalyzes the conversion of heme to iron, carbon monoxide and biliverdin employing oxygen and reducing equivalents. This enzyme is essential for heme-iron utilization and contributes to virulence in Leptospira interrogans. Methods: A phylogenetic analysis was performed using heme oxygenases sequences from different organisms including saprophytic and pathogenic Leptospira species. L. interrogans heme oxygenase (LepHO) was cloned, overexpressed and purified. The structural and enzymatic properties of LepHO were analyzed by UV–vis spectrophotometry and 1 H NMR. Heme-degrading activity, ferrous iron release and biliverdin production were studied with different redox partners. Results: A plastidic type, high efficiently ferredoxin-NADP+ reductase (LepFNR) provides the electrons for heme turnover by heme oxygenase in L. interrogans. This catalytic reaction does not require a ferredoxin. Moreover, LepFNR drives the heme degradation to completeness producing free iron and α-biliverdin as the final products. The phylogenetic divergence between heme oxygenases from saprophytic and pathogenic species supports the functional role of this enzyme in L. interrogans pathogenesis. Conclusions: Heme-iron scavenging by LepHO in L. interrogans requires only LepFNR as redox partner. Thus, we report a new substrate of ferredoxin-NADP+ reductases different to ferredoxin and flavodoxin, the only recognized protein substrates of this flavoenzyme to date. The results presented here uncover a fundamental step of heme degradation in L. interrogans. General significance: Our findings contribute to understand the heme-iron utilization pathway in Leptospira. Since iron is required for pathogen survival and infectivity, heme degradation pathway may be relevant for therapeutic applicationsFil: Soldano, Anabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Yao, Huili. University of Kansas; Estados UnidosFil: Rivera, Mario. University of Kansas; Estados UnidosFil: Ceccarelli, Eduardo Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Catalano Dupuy, Daniela Luján. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Biología Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario; Argentin

Redox Proteins as Targets for Drugs Development against Pathogens

Antimicrobial drug resistance in pathogens is an increasing human health problem. The rapid loss of effectiveness in antibiotics treatments and the accumulation of multi-resistant microbial strains are increasing worldwide threats. Moreover, several infectious diseases have been neglected for years and new antimicrobial treatments are lacking. In other cases, complexity of infectious organisms has exceeded the efforts to find new drugs to control them. Thus, strategies for the proper development of specific drugs are critically needed. Redox metabolism has already been proved to be a useful target for drug development. During the last years a significant number of electron carriers, enzymes, proteins and protein complexes have been studied and some of them were found to be essential for survival of several microbial pathogens. This review will focus on three major redox metabolic pathways which may provide promising strategies to fight against pathogens: the non-mevalonate pathway for isoprenoids biosynthesis, the iron metabolism and the iron-sulfur proteins. The common attractive link of all these processes is the plant-type ferredoxin-NADP+ reductase, an enzyme that participates in numerous electron transfer reactions and has no homologous enzyme in humans. Research in these redox pathways will open new perspectives for the rational design of drugs against infectious diseases.Fil: Catalano Dupuy, Daniela Luján. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Lopez Rivero, Arleth Susana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Soldano, Anabel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; ArgentinaFil: Ceccarelli, Eduardo Augusto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Rosario. Instituto de Biología Molecular y Celular de Rosario; Argentin

Small Molecule Inhibitors of the Bacterioferritin (BfrB)-Ferredoxin (Bfd) Complex Kill Biofilm-Embedded Pseudomonas aeruginosa Cells

© 2020 American Chemical Society. Bacteria depend on a well-regulated iron homeostasis to survive adverse environments. A key component of the iron homeostasis machinery is the compartmentalization of Fe3+ in bacterioferritin and its subsequent mobilization as Fe2+ to satisfy metabolic requirements. In Pseudomonas aeruginosa Fe3+ is compartmentalized in bacterioferritin (BfrB), and its mobilization to the cytosol requires binding of a ferredoxin (Bfd) to reduce the stored Fe3+ and release the soluble Fe2+. Blocking the BfrB-Bfd complex in P. aeruginosa by deletion of the bfd gene triggers an irreversible accumulation of Fe3+ in BfrB, concomitant cytosolic iron deficiency and significant impairment of biofilm development. Herein we report that small molecules developed to bind BfrB at the Bfd binding site block the BfrB-Bfd complex, inhibit the mobilization of iron from BfrB in P. aeruginosa cells, elicit a bacteriostatic effect on planktonic cells, and are bactericidal to cells embedded in mature biofilms

Reduction of the ferric LepHO-heme complex in anaerobic conditions and spontaneous reoxidation.

<p>Time dependent formation (A) and autoxidation (B) of the ferrous heme complex of wild type LepHO (●) and F157I mutant (○) as monitored by variations in absorbance at 426 and 403 nm, respectively. Data extracted from the spectra shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0182535#pone.0182535.g005" target="_blank">Fig 5</a>.</p