Identifying and targeting the Mg-Fe-PmrAB regulatory circuit reverses phosphate starvation-driven polymyxin resistance in Enterobacteriaceae

Phosphate (Pi) scarcity, a pervasive stressor prevalent in bacterial infections and dysbiotic host environments, potently drives antimicrobial resistance (AMR) against cationic antibiotics like polymyxins across diverse bacterial taxa. While established Pi depletion-induced AMR mechanisms often involve membrane phospholipid remodelling, Enterobacteriaceae largely lack these pathways, leaving their Pi scarcity-driven AMR mechanism unresolved. Here, we reveal that Pi limitation robustly induces polymyxin resistance in Enterobacteriaceae through 4-amino-4-deoxy-L-arabinose (L-Ara4N) modification of the Gram-negative outer membrane component lipid A, reflecting a distinct adaptive strategy. This modification is driven by strong ugd - arn operon induction, mediated by the PmrAB two-component system. We discover that Pi depletion triggers cellular Mg²⁺ release, prompting compensatory Fe³⁺ mobilization to the cell envelope that directly activates PmrAB. Crucially, this metal-dependent signaling axis offers a directly targetable mechanism for reversing polymyxin resistance, a significant deviation from the less accessible, PhoBR-regulated phospholipid remodelling strategies in other bacteria. We demonstrate that Mg²⁺ supplementation or Fe³⁺ chelation effectively suppresses PmrAB activation and restores polymyxin susceptibility, thereby establishing a novel, metal-centric paradigm for understanding and pharmacological intervention in stress-induced AMR.