Decoupling virulence from growth: a synthetic α-helical peptide suppresses Salmonella Typhimurium SPI-1 at sub-inhibitory concentrations

Cationic antimicrobial peptides (CAMPs) can alter bacterial physiology at concentrations below those required to inhibit growth, including modulation of virulence gene expression. In Salmonella Typhimurium, polymyxin B activates the PhoPQ envelope stress response and represses invasion-associated SPI-1 genes such as prgH , but whether synthetic CAMPs can selectively modulate virulence independently of antimicrobial activity remains unclear. Here, we evaluated a panel of rationally designed CAMPs, originally developed against Escherichia coli , for activity against Salmonella Typhimurium under physiological salt conditions used for in vitro induction of SPI-1. Among the peptides tested, the Leu/Ile-rich peptide L1L displayed the greatest antibacterial activity. In a real-time fluorescent assay, L1L also potently repressed prgH promoter activity at a concentration that had no measurable impact on growth kinetics, viability, or ultrastructure. Repression of prgH was accompanied by diminished invasion of epithelial cells in vitro . Relative to polymyxin B, L1L suppressed prgH expression at a smaller fraction of its MIC. Proteomic analysis revealed a coordinated response characterized by increased abundance of PhoPQ-associated CAMP resistance proteins, together with reduced abundance of SPI-1 regulators, structural proteins, effectors, motility proteins, and chemotaxis proteins. In contrast, the corresponding D-enantiomer produced a substantially weaker proteomic response, indicating that stereochemistry influences virulence modulation independently of antibacterial potency. These findings demonstrate that synthetic CAMPs can selectively reprogram Salmonella virulence at sub-inhibitory concentrations and identify L1L as a potent anti-virulence peptide that decouples suppression of host cell invasion from bacterial growth inhibition.