Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Soldano A

PROVIDER: S-EPMC7076691 | biostudies-literature | 2020 Mar

REPOSITORIES: biostudies-literature

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

Soldano Anabel A Yao Huili H Chandler Josephine R JR Rivera Mario M

ACS infectious diseases 20200115 3

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 Fe<sup ...[more]

PMID: 31898890

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Project description:Iron is an essential nutrient for bacteria but the reactivity of Fe2+ and the insolubility of Fe3+ present significant challenges to bacterial cells. Iron storage proteins contribute to ameliorating these challenges by oxidizing Fe2+ using O2 and H2O2 as electron acceptors, and by compartmentalizing Fe3+. Two types of iron-storage proteins coexist in bacteria, the ferritins (Ftn) and the heme-containing bacterioferritins (Bfr), but the reasons for their coexistence are largely unknown. P. aeruginosa cells harbor two iron storage proteins (FtnA and BfrB), but nothing is known about their relative contributions to iron homeostasis. Prior studies in vitro have shown that iron mobilization from BfrB requires specific interactions with a ferredoxin (Bfd), but the relevance of the BfrB:Bfd interaction to iron homeostasis in P. aeruginosa is unknown. In this work we explore the repercussions of (i) deleting the bfrB gene, and (ii) perturbing the BfrB:Bfd interaction in P. aeruginosa cells by either deleting the bfd gene or by replacing the wild type bfrB gene with a L68A/E81A double mutant allele in the P. aeruginosa chromosome. The effects of the mutations were evaluated by following the accumulation of iron in BfrB, analyzing levels of free and total intracellular iron, and by characterizing the ensuing iron homeostasis dysregulation phenotypes. The results reveal that P. aeruginosa accumulates iron mainly in BfrB, and that the nutrient does not accumulate in FtnA to detectable levels, even after deletion of the bfrB gene. Perturbing the BfrB:Bfd interaction causes irreversible flow of iron into BfrB, which leads to the accumulation of unusable intracellular iron while severely depleting the levels of free intracellular iron, which drives the cells to an acute iron starvation response despite harboring "normal" levels of total intracellular iron. These results are discussed in the context of a dynamic equilibrium between free cytosolic Fe2+ and Fe3+ compartmentalized in BfrB, which functions as a buffer to oppose rapid changes of free cytosolic iron. Finally, we also show that P. aeruginosa cells utilize iron stored in BfrB for growth in iron-limiting conditions, and that the utilization of BfrB-iron requires a functional BfrB:Bfd interaction.

Project description:The bfrB gene from Pseudomonas aeruginosa was cloned and expressed in Escherichia coli. The resultant protein (BfrB), which assembles into a 445.3 kDa complex from 24 identical subunits, binds 12 molecules of heme axially coordinated by two Met residues. BfrB, isolated with 5-10 iron atoms per protein molecule, was reconstituted with ferrous ions to prepare samples with a core mineral containing 600 +/- 40 ferric ions per BfrB molecule and approximately one phosphate molecule per iron atom. In the presence of sodium dithionite or in the presence of P. aeruginosa ferredoxin NADP reductase (FPR) and NADPH, the heme in BfrB remains oxidized, and the core iron mineral is mobilized sluggishly. In stark contrast, addition of NADPH to a solution containing BfrB, FPR, and the apo form of P. aeruginosa bacterioferritin-associated ferredoxin (apo-Bfd) results in rapid reduction of the heme in BfrB and in the efficient mobilization of the core iron mineral. Results from additional experimentation indicate that Bfd must bind to BfrB to promote heme mediation of electrons from the surface to the core to support the efficient mobilization of ferrous ions from BfrB. In this context, the thus far mysterious role of heme in bacterioferritins has been brought to the front by reconstituting BfrB with its physiological partner, apo-Bfd. These findings are discussed in the context of a model for the utilization of stored iron in which the significant upregulation of the bfd gene under low-iron conditions [Ochsner, U. A., Wilderman, P. J., Vasil, A. I., and Vasil, M. L. (2002) Mol. Microbiol. 45, 1277-1287] ensures sufficient concentrations of apo-Bfd to bind BfrB and unlock the iron stored in its core. Although these findings are in contrast to previous speculations suggesting redox mediation of electron transfer by holo-Bfd, the ability of apo-Bfd to promote iron mobilization is an economical strategy used by the cell because it obviates the need to further deplete cellular iron levels to assemble iron-sulfur clusters in Bfd before the iron stored in BfrB can be mobilized and utilized.

Project description:The structure of recombinant Pseudomonas aeruginosa bacterioferritin B (Pa BfrB) has been determined from crystals grown from protein devoid of core mineral iron (as-isolated) and from protein mineralized with approximately 600 iron atoms (mineralized). Structures were also obtained from crystals grown from mineralized BfrB after they had been soaked in an FeSO(4) solution (Fe soak) and in separate experiments after they had been soaked in an FeSO(4) solution followed by a soak in a crystallization solution (double soak). Although the structures consist of a typical bacterioferritin fold comprised of a nearly spherical 24-mer assembly that binds 12 heme molecules, comparison of microenvironments observed in the distinct structures provided interesting insights. The ferroxidase center in the as-isolated, mineralized, and double-soak structures is empty. The ferroxidase ligands (except His130) are poised to bind iron with minimal conformational changes. The His130 side chain, on the other hand, must rotate toward the ferroxidase center to coordinate iron. In comparison, the structure obtained from crystals soaked in an FeSO(4) solution displays a fully occupied ferroxidase center and iron bound to the internal, Fe((in)), and external, Fe((out)), surfaces of Pa BfrB. The conformation of His130 in this structure is rotated toward the ferroxidase center and coordinates an iron ion. The structures also revealed a pore on the surface of Pa BfrB that likely serves as a port of entry for Fe(2+) to the ferroxidase center. On its opposite end, the pore is capped by the side chain of His130 when it adopts its "gate-closed" conformation that enables coordination to a ferroxidase iron. A change to its "gate-open", noncoordinative conformation creates a path for the translocation of iron from the ferroxidase center to the interior cavity. These structural observations, together with findings obtained from iron incorporation measurements in solution, suggest that the ferroxidase pore is the dominant entry route for the uptake of iron by Pa BfrB. These findings, which are clearly distinct from those made with Escherichia coli Bfr [Crow, A. C., Lawson, T. L., Lewin, A., Moore, G. R., and Le Brun, N. E. (2009) J. Am. Chem. Soc. 131, 6808-6813], indicate that not all bacterioferritins operate in the same manner.

Dataset Information

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

Publications

Inhibiting Iron Mobilization from Bacterioferritin in <i>Pseudomonas aeruginosa</i> Impairs Biofilm Formation Irrespective of Environmental Iron Availability.

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