Iron-responsive phosphorylation of TolQ modulates cell envelope integrity and antibiotic susceptibility in Klebsiella pneumoniae

Abstract Klebsiella pneumoniae is an opportunistic bacterial pathogen associated with high morbidity and mortality, exacerbated by the rapid emergence of resistance to last-resort antibiotics, such as carbapenems. Adaptation to nutrient limitation, particularly fluctuations in metal availability, is critical for bacterial survival and virulence, yet the regulatory mechanisms coordinating these responses remain incompletely understood. Protein phosphorylation represents a key post-translational modification governing bacterial physiology and offers a promising avenue for identifying novel antimicrobial targets. Here, we applied mass spectrometry–based phosphoproteomics to define nutrient-responsive signaling networks in K. pneumoniae under varying iron and zinc conditions. This analysis identified iron-dependent phosphorylation of TolQ, a conserved inner membrane component of the Tol–Pal system that maintains cell envelope integrity. Structural modeling predicted that phosphorylation modulates TolQ–TolR conformation, suggesting a mechanism by which iron availability regulates Tol–Pal function. Functional characterization demonstrated that deletion of tolQ results in reduced bacterial viability, increased susceptibility to host immune clearance, and heightened sensitivity to antibiotic treatment. To further explore the therapeutic potential of this pathway, we integrated high-throughput compound screening with computational modeling and identified small molecules that phenocopy Δ tolQ . Collectively, these findings reveal a previously unrecognized link between iron availability and phosphoregulation of the Tol–Pal system and establish TolQ as a critical mediator of bacterial survival. This work highlights phosphoproteomics as a powerful strategy to uncover regulatory vulnerabilities and identify targets for antimicrobial development in drug-resistant pathogens.