Small Molecule Inhibitors of the BfrB-Bfd Interaction Decrease Pseudomonas aeruginosa Fitness and Potentiate Fluoroquinolone Activity.

The iron storage protein bacterioferritin (BfrB) is central to bacterial iron homeostasis. The mobilization of iron from BfrB, which requires binding by a cognate ferredoxin (Bfd), is essential to the regulation of cytosolic iron levels in P. aeruginosa. This paper describes the structure-guided development of small molecule inhibitors of the BfrB-Bfd protein-protein interaction. The process was initiated by screening a fragment library and followed by obtaining the structure of a fragment hit bound to BfrB. The structural insights were used to develop a series of 4-(benzylamino)- and 4-((3-phenylpropyl)amino)-isoindoline-1,3-dione analogs that selectively bind BfrB at the Bfd binding site. Challenging P. aeruginosa cells with the 4-substituted isoindoline analogs revealed a dose-dependent growth phenotype. Further investigation determined that the analogs elicit a pyoverdin hyperproduction phenotype that is consistent with blockade of the BfrB-Bfd interaction and ensuing irreversible accumulation of iron in BfrB, with concomitant depletion of iron in the cytosol. The irreversible accumulation of iron in BfrB prompted by the 4-substituted isoindoline analogs was confirmed by visualization of BfrB-iron in P. aeruginosa cell lysates separated on native PAGE gels and stained for iron with Ferene S. Challenging P. aeruginosa cultures with a combination of commercial fluoroquinolone and our isoindoline analogs results in significantly lower cell survival relative to treatment with either antibiotic or analog alone. Collectively, these findings furnish proof of concept for the usefulness of small molecule probes designed to dysregulate bacterial iron homeostasis by targeting a protein-protein interaction pivotal for iron storage in the bacterial cell.

Inhibiting the BfrB:Bfd interaction in Pseudomonas aeruginosa causes irreversible iron accumulation in bacterioferritin and iron deficiency in the bacterial cytosol.

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.

Antibiotic activity of iron-sequestering polymers.

Increasing antibiotic resistance has compelled the development of novel antibiotics and adjuvant therapies that enhance the efficacy of existing antibiotics. Iron plays a critical role in bacterial infections, yet the use of iron chelators as adjuvant therapy with antibiotics has yielded highly variable outcomes. Multivalent polymeric materials offer an alternative approach to bind and sequester iron via high avidity interactions. Here, a biomimetic iron-sequestering polymer (PAI-DHBA) was synthesized by modifying side chains of cross-linked polyallylamine (cPAI) with 2,3-dihydroxybenzoic acid (DHBA). PAI-DHBA polymer gels with various DHBA contents showed high iron affinity indices and high selectivity for iron. The polymers showed mild antibiotic properties when used to treat established bacterial cultures. Pretreating culture media with PAI-DHBA polymer, however, removed all detectable iron from media and effectively inhibited the growth of Pseudomonas aeruginosa. In addition, bacterial growth was more susceptible to antibiotics combined with PAI-DHBA. Multivalent polymers that bind and sequester iron, such as PAI-DHBA, offer a promising early intervention or adjuvant to antibiotics.

Concerted motions networking pores and distant ferroxidase centers enable bacterioferritin function and iron traffic.

X-ray crystallography, molecular dynamics (MD) simulations, and biochemistry were utilized to investigate the effect of introducing hydrophobic interactions in the 4-fold (N148L and Q151L) and B-pores (D34F) of Pseudomonas aeruginosa bacterioferritin B (BfrB) on BfrB function. The structures show only local structural perturbations and confirm the anticipated hydrophobic interactions. Surprisingly, structures obtained after soaking crystals in Fe2+-containing crystallization solution revealed that although iron loads into the ferroxidase centers of the mutants, the side chains of ferroxidase ligands E51 and H130 do not reorganize to bind the iron ions, as is seen in the wt BfrB structures. Similar experiments with a double mutant (C89S/K96C) prepared to introduce changes outside the pores show competent ferroxidase centers that function akin to those in wt BfrB. MD simulations comparing wt BfrB with the D34F and N148L mutants show that the mutants exhibit significantly reduced flexibility and reveal a network of concerted motions linking ferroxidase centers and 4-fold and B-pores, which are important for imparting ferroxidase centers in BfrB with the required flexibility to function efficiently. In agreement, the efficiency of Fe2+ oxidation and uptake of the 4-fold and B-pore mutants in solution is significantly compromised relative to wt or C89S/K96C BfrB. Finally, our structures show a large number of previously unknown iron binding sites in the interior cavity and B-pores of BfrB, which reveal in unprecedented detail conduits followed by iron and phosphate ions across the BfrB shell, as well as paths in the interior cavity that may facilitate nucleation of the iron phosphate mineral.