FhuA-SbmA-driven resistance pathway to microcin Y triggers transcriptomic reprogramming and virulence enhancement in Salmonella Typhimurium.

Salmonella Typhimurium poses critical safety risks owing to its multidrug resistance and persistence in food matrices. To address this threat, we focused on microcin Y (MccY) a lasso peptide with potent activity against multidrug-resistant S. typhimurium and elucidated how mutations in the FhuA/SbmA receptors mediate MccY resistance and differentially affect bacterial virulence. Molecular docking revealed that MccY tended to bind to the N-terminal segment of FhuA within its β-barrel cavity, with key interaction sites including FhuA564Leu-MccY5His (2.5 Å hydrogen bond, MIC = 200 μg/mL) and sites such as FhuA129Ser/FhuA147Asn (MIC = 100-200 μg/mL). For SbmA, MccY forms hydrogen bonds with SbmA204Asn-MccY20Tyr (3.1 Å, MIC = 1.0 μg/mL), SbmA320Asn-MccY21Gly (2.9 Å, MIC = 200 μg/mL), and SbmA361Gln-MccY21Gly (2.9 Å, MIC = 2.0 μg/mL). The susceptibility of MccY's lasso structure domains to resistance was ranked as follows: tail < ring < loop. FhuA/SbmA mutations enhance MccY resistance (MIC>250 μg/mL) via receptor conformational changes, with FhuAAla63Gly, and FhuA401Met402Arg conferring higher resistance than SbmAGly254Glu, and SbmAGln361Leu. Chrome Azure S assays confirmed that these mutations, particularly FhuA401Met402Arg insertions and SbmAGly254Glu, and SbmAGln361Leu substitutions, disrupt iron transport. Notably, FhuA mutants exhibited altered siderophore utilization, extracellular iron accumulation, enhanced biofilm formation (p < 0.05), increased flagellar motility (migration 1.7-2.7 cm), and attenuated virulence. In contrast, SbmA mutants showed broader metabolic remodeling and downregulated invasive virulence genes (prgI, invE), which correlates with enhanced in vivo virulence in mice. FhuA/SbmA mutations in S. typhimurium drive resistance, adaptive fitness, and pathogenicity through defined receptor-ligand interactions. This work provides a molecular framework for developing integrated antimicrobial approaches to mitigate the spread of resistant pathogens.