Decoding growth inhibitory associated pathways of xenometal–siderophore antibiotic conjugates in <i>S. aureus</i>

Pathogenic <i>Staphylococcus aureus</i> causes most infectious disease related deaths in the developed world. Continuously evolving resistance to clinically approved antibiotics and combination therapies limits treatment 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 efficacy. 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 gallium xenometallomycins (siderophore antibiotic conjugates incorporating non-endogenous metal ions), Ga-DFO-Cip and Ga-LDFC-Cip, which have demonstrated high potency compared to the parent antibiotic's efficacy <i>in vitro</i> in <i>S. aureus</i> infection. 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 in <i>S. aureus</i> bacterial strains. 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 <i>S. aureus</i> strains is impaired, leading to diminished compound accumulation <i>in vitro</i> and <i>in vivo</i>.