Pathogenic Staphylococcus aureus causes most infectious disease related deaths in the developed world. Continuously evolving resistance to clinically approved antibiotics and combination therapies limits tratment efficacy; new strategies that evade and slow resistance, or produce resistant mutants with reduced fitness are needed. We employ antibiotics conjugated to bacterially recognized siderophores to potentiate their effiacy. Acting as a Trojan Horse, the siderophore antibiotic conjugates efficiently deliver the antibiotic inside the bacterial cytoplasm by hijacking the iron transport system pathways which are crucial for bacterial survival. Here, we investigated the mechanism of action of the gallium xenometallomycins (siderophore antibiotic conjugates incorporating non-endogenous metal ions) Ga-DFO-Cip and Ga-LDFC-Cip, that have demonstrated high potency compared to the parent antibiotic’s efficacy in vitro. Employing physicochemical, synthetic and transcriptomic analysis studies, this work reveals that kinetically inert, gallium-containing xenometallomycins targeting cytoplasmic bacterial targets impart differential resistance and gene expression profiles when compared to their parent antibiotic. Both Ga-DFO-Cip and Ga-LDFC-Cip effectively disrupt iron-siderophore biosynthesis and uptake machinery. We affirm our results with the radioactive surrogate 67/68Ga-DFO-Cip and demonstrate that the bacterial uptake in Ga-DFO-Cip-resistant strains is impaired, leading to diminished compound accumulation in vitro and in vivo.
