LuxS/AI-2 regulates phoP/phoQ by a non-canonical mechanism to enhance acid stress survival in Salmonella Typhimurium

The intestinal milieu is largely characterized by the complex array of chemical compounds produced through the metabolic activity of resident microbiota. Enteric pathogens like Salmonella, which have evolved refined mechanisms to persist within this environment, utilize these microbial metabolites and self-produce quorum molecules as molecular cues to identify ecological niches and modulate their survival and virulence strategies. Salmonella quorum sensing involves producing and detecting universal Autoinducer-2 (AI-2) signaling molecules. Our research reveals that Salmonella Typhimurium enhances AI-2 biosynthesis and transport under acidic conditions, aiding environmental adaptation and facilitating pathogenesis in macrophages. AI-2 signaling regulates the pH-sensing two-component system genes, phoP/phoQ, ensuring cytosolic pH homeostasis, survival, and acid tolerance. It also involves regulating the lysine/cadaverine-mediated acid tolerance response and maintaining bacterial cytosolic pH. Furthermore, we investigated the mechanism of AI-2-mediated gene regulation and demonstrated that, in addition to the lsr promoter, the repressor LsrR binds the phoP promoter via its Y25 and R43 residues, thereby negatively regulating phoP expression. Additionally, this signaling ameliorates the intracellular survival by modulating Salmonella Pathogenicity Island-2 (SPI-2) regulators (ssrA/ssrB) and SPI-2 effector expression via PhoP. Mouse models demonstrate that AI-2 signaling is essential for colonizing the primary and secondary infection sites. Therefore, quorum sensing facilitates survival in hostile host environments by modulating multiple genetic targets through the AI-2/LsrR-mediated feedback loop in pathogenic bacteria.Author summary: To survive and cause disease within the host, enteric pathogens such as Salmonella sense and respond to changing environmental cues. One key strategy is quorum sensing, a cell–cell communication system. Salmonella uses the conserved quorum-sensing signal autoinducer-2 (AI-2) to coordinate adaptation and virulence. In this study, we show that Salmonella Typhimurium increases both AI-2 production and uptake under acidic conditions. AI-2 signaling promotes adaptation to hostile host environments by maintaining cytosolic pH homeostasis and enhancing survival inside macrophages. We demonstrate that AI-2 regulates major stress-response pathways, including the PhoP/PhoQ two-component system, and controls acid tolerance mechanisms involving lysine and cadaverine metabolism. We further identify a direct mechanism of AI-2–mediated gene regulation. The AI-2 responsive repressor LsrR binds not only to its canonical target but also to the phoP promoter, thereby fine-tuning stress responses. Through this regulatory network, AI-2 signaling modulates the expression of Salmonella Pathogenicity Island-2 genes required for intracellular survival. Using mouse infection models, we show that AI-2 signaling is essential for efficient colonization of both primary and secondary infection sites. Together, these findings establish quorum sensing as a central mechanism enabling Salmonella to survive hostile host environments and establish infection.