Siderophores are natural products commonly produced by bacteria and fungi. They act as iron(III)-chelating agents that are secreted outside of the cell in environments with low bioavailable iron concentrations. Siderophores have extraordinarily high iron-binding affinities, allowing them to scavenge metal from many environmental and biological sources (i.e. transferrin). These siderophores are used clinically to treat iron overload disease, and have promising activity against other human diseases such as malaria and cancer. In gram-positive bacteria, siderophore-binding proteins (SBP) are membrane-anchored lipoproteins displayed on the cell surface to bind and facilitate entry of siderophores into the cell. FhuD2 is a SBP produced by human pathogenic Staphylococcus aureus that has broad specificity towards binding trihydroxamate sideophores. This work focuses on a new affinity chromatography method to isolate ferrioxamine siderophores from complex mixtures by utilizing Ni-NTA resin-immobilized FhuD2. Dilute solutions containing a ferrioxamine siderophore are passed through immobilized FhuD2 and are subsequently eluted by the addition of a sacrificial siderophore that displaces the siderophore of interest from the SBP-resin. The SBP-resin enabled purification of charge positive, charge negative, and charge neutral ferrioxamine siderophores allowing for flexibility in the choice of eluting siderophore to facilitate removal by simple ion exchange chromatography. To demonstrate utility for isolating siderophores from complex mixtures, culture supernatants from Streptomyces violaceus DSM 8286 were treated with FhuD2 SBP-resin, which enabled isolation of the salmycins, a mixture of glycosylated sideromycins (siderophore-antibiotic conjugate) with potent antibacterial activity toward human pathogenic Staphylococcus aureus. The FhuD2 SBP-resin can be reused in iterative cycles of siderophore sequestration and elution to enable preparative scale isolation of siderophores and sideromycins, which greatly simplifies the purification of these natural products that are typically produced at very low concentrations from microbial cultures. This work represents an enabling technology for siderophore-affinity chromatography that could be interfaced with genomic and metabolomic natural product discovery platforms to enhance isolation of new siderophore and sideromycin natural products in a biased or unbiased manner based on the choice of SBP-resin.

Immobilized FhuD2 siderophore-binding protein enables purification of salmycin sideromycins from Streptomyces violaceus DSM 8286

256th National Meeting (2018)

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