Human-targeted drugs extensively reshape regulatory networks controlling virulence and antibiotic resistance in Salmonella Typhimurium

Abstract Human-targeted drugs are increasingly recognized to affect bacterial physiology, yet their impact beyond growth inhibition, and the underlying molecular mechanisms, remain poorly understood. Here, we systematically mapped how chemically diverse host-associated compounds influence virulence and intrinsic antibiotic resistance in Salmonella enterica serovar Typhimurium. Using a high-throughput reporter approach, we quantified transcriptional response of 26 key stress promoters in Salmonella against 2,415 compounds, including FDA-approved drugs, food-derived molecules, and human metabolites. We found that non-antibiotic compounds induce transcriptional responses distinct from those elicited by antibiotics, uncovering a regulatory shift from general stress responses to compound-specific regulation driven by ligand-responsive MarR- and TetR-family regulators. In particular, more than 200 non-antibiotic drugs modulate virulence or intrinsic resistance pathways through the central regulators SlyA or RamR, respectively. By integrating our large-scale dataset with machine learning, we further defined chemical features associated with ramA transcriptional activation in Salmonella , and predicted ∼200 additional candidate inducers of intrinsic antibiotic resistance in this pathogen. Together, our results uncovered a large and previously unrecognized chemical landscape that reshapes bacterial stress responses through ligand-responsive regulatory mechanisms, with implications for how host-associated compounds influence pathogen virulence and antibiotic resistance. Highlights Mapping the transcriptional response of key Salmonella stress pathways across a large chemical space shows that antibiotics and non-antibiotic compounds elicit distinct stress responses in Salmonella The activity of the virulence regulator SlyA is modulated by non-antibiotic compounds SlyA-inhibitors increase sensitivity to polymyxin B and decrease Salmonella virulence in cell culture Human-targeted drugs increase intrinsic antibiotic resistance via RamR-RamA in Salmonella Machine learning approach enables large-scale, out-of-sample prediction of human-targeted drugs inducing ramA expression in Salmonella