Immobilized FhuD2 Siderophore-Binding Protein Enables Purification of Salmycin Sideromycins from Streptomyces violaceus DSM 8286.

Siderophores are a structurally diverse class of natural products common to most bacteria and fungi as iron(III)-chelating ligands. Siderophores, including trihydroxamate ferrioxamines, are used clinically to treat iron overload diseases and show promising activity against many other iron-related human diseases. Here, we present a new method for the isolation of ferrioxamine siderophores from complex mixtures using affinity chromatography based on resin-immobilized FhuD2, a siderophore-binding protein (SBP) from Staphylococcus aureus. The SBP-resin enabled purification of charge positive, charge negative, and neutral ferrioxamine siderophores. Treatment of culture supernatants from Streptomyces violaceus DSM 8286 with SBP-resin provided an analytically pure sample of the salmycins, a mixture of structurally complex glycosylated sideromycins (siderophore-antibiotic conjugates) with potent antibacterial activity toward human pathogenic Staphylococcus aureus (minimum inhibitory concentration (MIC) = 7 nM). Siderophore affinity chromatography could enable the rapid discovery of new siderophore and sideromycin natural products from complex mixtures to aid drug discovery and metabolite identification efforts in a broad range of therapeutic areas.

Structural Basis for Xenosiderophore Utilization by the Human Pathogen Staphylococcus aureus.

Staphylococcus aureus produces a cocktail of metallophores (staphylopine, staphyloferrin A, and staphyloferrin B) to scavenge transition metals during infection of a host. In addition, S. aureus displays the extracellular surface lipoproteins FhuD1 and FhuD2 along with the ABC transporter complex FhuCBG to facilitate the use of hydroxamate xenosiderophores such as desferrioxamine B (DFOB) for iron acquisition. DFOB is used as a chelation therapy to treat human iron overload diseases and has been linked to an increased risk of S. aureus infections. We used a panel of synthetic DFOB analogs and a FhuD2-selective trihydroxamate sideromycin to probe xenosiderophore utilization in S. aureus and establish structure-activity relationships for Fe(III) binding, FhuD2 binding, S. aureus growth promotion, and competition for S. aureus cell entry. Fe(III) binding assays and FhuD2 intrinsic fluorescence quenching experiments revealed that diverse chemical modifications of the terminal ends of linear ferrioxamine siderophores influences Fe(III) affinity but not FhuD2 binding. Siderophore-sideromycin competition assays and xenosiderophore growth promotion assays revealed that S. aureus SG511 and ATCC 11632 can distinguish between competing siderophores based exclusively on net charge of the siderophore-Fe(III) complex. Our work provides a roadmap for tuning hydroxamate xenosiderophore scaffolds to suppress (net negative charge) or enhance (net positive or neutral charge) uptake by S. aureus for applications in metal chelation therapy and siderophore-mediated antibiotic delivery, respectively.