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 <i><i>Escherichia coli</i></i> and <i>Salmonella enterica</i> 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, <i><i>Pseudomonas aeruginosa</i></i> and <i><i>Acinetobacter baumannii</i></i>, which use Ent as a xeno-siderophore. We uncover the ability of <i><i>A. baumannii</i></i> 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.