Iron sequestration is critical for bacterial viability, driving the evolution of elaborate mechanisms for iron acquisition. These include siderophores, high-affinity iron chelators, and dedicated import pathways for iron-siderophore complexes.1 Siderophore-antibiotic conjugates exploit the inherent iron uptake machinery of bacteria, essentially functioning as Trojan horses to deliver antibiotics intracellularly.2 Our research, along with others’, demonstrates that gallium-based siderophore-antibiotic conjugates significantly enhance the activity of broad-spectrum antibiotics. However, the import mechanism remains elusive. 3,4 This knowledge gap is partly due to limitations in identifying the responsible trans-membrane transporters, whose discovery currently relies on laborious mutant generation and growth analysis.5 To address this, novel tools are needed to expedite transporter discovery and characterization.
Modular deferoxamine (DFO)-based photoaffinity probes were designed to identify transmembrane and associated proteins involved in xenometal siderophore uptake. These probes were strategically incorporated with a photoactive diazirine moiety, allowing for light-induced covalent crosslinking to target proteins. Additionally, the probes were equipped with fluorophore for visualization of labeled proteins and biotin for enrichment and identification. This modular design offers straightforward solid-phase synthesis and efficient library generation.
This work focuses on the development, optimization, and validation of the M-DFO-azir-0X ( M= apo, Ga, or Fe) photoprobe series for the identification of the FoxA transmembrane deferoxamine transporter endogenous to P. aeruginosa. Screening of single amino acids with these probes revealed preferential reactivity towards aspartic acid and tyrosine residues. Notably, the co-crystal structure of FoxA bound to Fe-DFO-azir-01 provides compelling evidence that FoxA binds the photoaffinity probe in a manner analogous to Fe-DFO. MS/MS analysis employing purified FoxA protein tagged with Fe-DFO-azir-02 corroborated labeling of the diazirine moiety within the binding pocket, specifically targeting tyrosine residues. Live-cell labeling experiments utilizing M-DFO-azir-05 in a FoxA-overexpressing E. coli lemo21 mutant strain demonstrated successful probe uptake and in vivo protein labeling. MS/MS analysis of the fluorescent band confirmed the tagging of FoxA.
In conclusion, we have successfully optimized DFO-based photoaffinity probes to target the FoxA siderophore receptor in bacteria. Further enrichment assays using M-DFO-azir-06 will be employed to identify other tagged proteins visualized by gel fluorescence imaging. Additionally, we will investigate the influence of iron availability within the culture medium on the protein tagging profile in E.coli K-12. This investigation will provide valuable insights into dynamics of probe-protein interactions under different iron conditions.
Image/Figure:
Click to view full size
Image/Figure Caption:
A cartoon outlining the steps of siderophore-photoaffinity probes, from labeling proteins within a living organism.
Author
Graduate student
University of Wisconsin Madison