Pseudomonas aeruginosa FpvB Is a High-Affinity Transporter for Xenosiderophores Ferrichrome and Ferrioxamine B

Iron is essential for many biological functions in bacteria, but its poor solubility is a limiting factor for growth. Bacteria produce siderophores, soluble natural products that bind iron with high affinity, to overcome this challenge. Siderophore-iron complexes return to the cell through specific outer membrane transporters. The opportunistic pathogen Pseudomonas aeruginosa makes multiple transporters that recognize its own siderophores, pyoverdine and pyochelin, and xenosiderophores produced by other bacteria or fungi, which gives it a competitive advantage. Some antibiotics exploit these transporters to bypass the membrane to reach their intracellular targets—including the thiopeptide antibiotic, thiostrepton (TS), which uses the pyoverdine transporters FpvA and FpvB to cross the outer membrane. Here, we assessed TS susceptibility in the presence of various siderophores and discovered that ferrichrome and ferrioxamine B antagonized TS uptake via FpvB. Unexpectedly, we found that FpvB transports ferrichrome and ferrioxamine B with higher affinity than pyoverdine. Site-directed mutagenesis of FpvB coupled with competitive growth inhibition and affinity label quenching studies suggested that the siderophores and antibiotic share a binding site in an aromatic pocket formed by the plug and barrel domains but have differences in their binding mechanism and molecular determinants for uptake. This work describes an alternative uptake pathway for ferrichrome and ferrioxamine B in P. aeruginosa and emphasizes the promiscuity of siderophore transporters, with implications for Gram-negative antibiotic development via the Trojan horse approach.