Siderophore-Pt(IV) Conjugates as Tools to Probe Cytoplasmic Cargo Delivery to Gram-Negative Bacteria

Elucidating the uptake pathways and cellular localization of bacterial siderophores is important for establishing a fundamental understanding of Fe homeostasis and trafficking. Discerning the same for nonnative siderophores is a key consideration in the translational development of siderophore-antibiotic conjugates for "Trojan-horse" drug delivery. Here, we employ the native triscatecholate siderophore enterobactin (Ent) or its synthetic mimic TRENCAM (TC) conjugated to a Pt(IV) prodrug based on cisplatin to study the localization of these siderophores within various Gram-negative bacteria. The Pt(IV) warhead, which is reductively activated upon delivery to the cytoplasm, targets DNA and provides the readout of filamentous morphology, repurposing the anticancer drug as a probe for bacterial cytoplasmic delivery. By applying TC-Pt(IV) as a delivery probe, we establish that uropathogenic Escherichia coli and Salmonella enterica serovar Typhimurium transport TC to the cytoplasm via FepCDG, the inner membrane transport machinery for Ent. Furthermore, we use Ent-Pt(IV) and TC-Pt(IV) to study the cellular localization of triscatecholate siderophores in two ESKAPE pathogens, Pseudomonas aeruginosa and Acinetobacter baumannii, which use Ent as a xeno-siderophore. We uncover the ability of A. baumannii to transport Ent and TC to the cytoplasm and carry out bioinformatic analyses to identify a putative inner membrane transport system. Utilizing siderophore-Pt(IV) prodrug conjugates as probes for cellular localization, our findings provide insights into the location and specificity of siderophore transport machinery, particularly toward nonnative and xeno-siderophores, while illuminating the potential of siderophore-antibiotic conjugates based on Ent or TC for the delivery of cytoplasmic targeting antibiotics in multiple Gram-negative bacterial pathogens.