IRON-AMR

Antibiotic resistance is a growing threat to public health, particularly concerning Gram-negative bacteria, which are more difficult to treat due to their protective outer membrane. This membrane limits antibiotic entry, making Gram-negative bacteria more resistant to treatments. While it has long been believed that antibiotics enter Gram-negative bacteria primarily through porins, recent studies have challenged this assumption, revealing that many antibiotics use alternative mechanisms to cross the outer membrane. One such mechanism may involve TonB-dependent transporters (TBDTs), which are known to facilitate the uptake of nutrients such as iron-siderophore complexes. Additionally, efflux systems, which expel antibiotics from the cell, contribute to resistance by reducing intracellular drug concentrations. Several studies have highlighted an interaction between iron availability and antibiotic efficacy. Iron is a vital nutrient for bacterial survival but is scarce during infection, as the host employs a strategy known as nutritional immunity to restrict its availability. Our previous results have shown that iron deficiency can alter the composition of the outer membrane, increasing the expression of TBDTs and certain efflux systems, which in turn affects membrane permeability. However, antibiotic susceptibility testing in the pharmaceutical industry and clinical settings is often conducted under iron-rich conditions that do not reflect the iron-restricted environments encountered during infection. The IRON-AMR project aims to explore the relationship between bacterial iron acquisition and antibiotic efficacy in the pathogen Pseudomonas aeruginosa, focusing on two main aspects: (1) antibiotic import via TBDTs and (2) their efflux via PvdRT-OpmQ and MexAB-OprM. We hypothesize that iron restriction alters the expression of these transporters and efflux pumps, thereby influencing antibiotic susceptibility. The project will use fluorescent reporters and proteomics to study TBDT and efflux pump expression in P. aeruginosa grown under iron-restricted conditions. Deletion mutants and screening methods will identify which antibiotics are imported via TBDTs and assess how TBDT expression impacts antibiotic activity. The contribution of PvdRT-OpmQ and MexAB-OprM efflux systems to antibiotic efflux under iron-restricted conditions will also be explored. Semi-mechanistic pharmacokinetic/pharmacodynamic (PK/PD) models will be used to quantify resistance and bacterial growth under iron-restricted conditions, helping to evaluate how iron-restricted media affect antibiotic efficacy. While P. aeruginosa will serve as the primary model organism, the findings obtained from P. aeruginosa will be extended to other pathogens, such as Salmonella enterica and Klebsiella pneumoniae, to assess the broader implications of iron restriction on antibiotic susceptibility. Additionally, the project will also validate whether PvdRT-OpmQ and MexAB-OprM transport antibiotics through in vitro efflux assays using these systems reconstituted in biomimetic membrane environments. In silico modeling will also be used to study the interactions between these efflux pumps and their substrates. This project will involve three partners with expertise in iron homeostasis, microbiology, PK/PD modeling, and the biochemistry and structural biology of membrane proteins. The originality of IRON-AMR lies in its exploration of the interplay between iron availability and antibiotic efficacy, an underexplored area in antimicrobial research. By focusing on TBDTs and efflux pumps under iron-restricted conditions, the project aims to elucidate poorly understood mechanisms of antibiotic resistance. This project will enhance our understanding of antibiotic import and export under infection-like conditions, addressing critical gaps in current testing methods and opening new avenues for more effective treatments.