bioRxiv2026
Multidrug-resistant (MDR) Pseudomonas aeruginosa poses a significant clinical challenge due to its poorly permeable outer membrane, efflux systems, biofilm formation, and rapid acquisition of resistance genes. The lack of new treatments for P. aeruginosa infections underscores the necessity for innovative therapeutic solutions. Iron uptake is essential for bacterial survival, making it a promising target for the development of new antimicrobials. Iron-chelating siderophores are vital for bacterial iron acquisition, important agents for direct antimicrobial action, adjuvants to enhance the effectiveness of currently available antibiotics, and components of prodrugs that facilitate the transport of covalently linked antibiotics into the cell. Here, we report the anti-pseudomonal activity of vacidobactin A, a siderophore produced by the soil bacterium Variovorax paradoxus, identified through a screen of natural product extracts targeting a clinical MDR strain of P. aeruginosa. Vacidobactin A inhibits P. aeruginosa growth by limiting iron availability, particularly in strains that do not produce pyoverdine, their native siderophore. Expression of a TonB-dependent transporter sourced from the vacidobactin producer in a P. aeruginosa pyoverdine and pyochelin-null mutant restored its ability to acquire iron and grow in the presence of vacidobactin. Additionally, vacidobactin A synergized with thiostrepton, which hijacks pyoverdine receptors to enter the cell and inhibit protein synthesis. This study supports the therapeutic potential of targeting P. aeruginosa iron acquisition pathways and leveraging siderophores as adjuvants to enhance the efficacy of existing antimicrobials. These findings, along with recent advancements in siderophore-based research and combination therapies, offer innovative strategies to combat antibiotic-resistant infections. IMPORTANCEMultidrug-resistant Pseudomonas aeruginosa is a critical priority pathogen for which new therapeutic strategies are urgently needed. Iron acquisition is essential for P. aeruginosa survival and virulence, yet remains underexploited as a drug target. Here, we demonstrate that vacidobactin A, a siderophore produced by Variovorax paradoxus, suppresses P. aeruginosa growth by limiting iron availability, particularly in strains deficient in pyoverdine production. We further show that vacidobactin A enhances the activity of thiostrepton, an antibiotic that exploits siderophore uptake pathways. These findings highlight iron competition as a source of anti-pseudomonal agents and support the development of siderophore-based therapeutics and adjuvant strategies. Targeting iron acquisition networks offers a mechanistically distinct approach to combat antibiotic resistance and expands the repertoire of vulnerabilities that can be leveraged against this highly drug-resistant pathogen.