Inhibiting Bacterioferritin Iron Release Induces Iron Starvation and Metabolic Downshift, Potentiating...

In Pseudomonas aeruginosa, bacterioferritin contributes to regulating cytosolic iron by oxidizing Fe²⁺ and storing Fe³⁺ within its internal cavity, and binding to its cognate ferredoxin to reduce stored Fe³⁺ and release Fe²⁺ to the cytosol. Small molecule derivatives of 4-aminoisoindoline-1,3-dione bind P. aeruginosa bacterioferritin at the cognate ferredoxin binding site, inhibit iron release from bacterioferritin, and exhibit bactericidal activity against mature P. aeruginosa biofilms. In this study we report that treatment of mature biofilms with the 4-aminoisoindoline-1,3-dione derivative KM-5-35 induced approximately 90% cell death. Proteomic analysis of surviving cells revealed iron limitation accompanied by disrupted central carbon metabolism, reduced fatty acid and amino acid biosynthesis, and alterations in ribosome protein composition. These metabolic downshifts likely resulted in unstable proton motive force, diminished efflux capacity and impaired ribosome turnover. In this compromised state, gentamicin and amikacin, but not tobramycin, efficiently exploit KM-5-35-induced translational stress, leading to profound synergistic killing of biofilm cells. These findings underscore the therapeutic potential of targeting bacterioferritin iron mobilization in biofilm-associated infections and suggest a promising strategy for combination antimicrobial therapy.