FLIM of mCherryTYG Deciphers pH Dynamics and Lifestyles of <i>Salmonella</i> Typhimurium

Intracellular pH regulation is fundamental to bacterial adaptation, virulence, and survival in diverse environments. <i>Salmonella</i> Typhimurium, a key human pathogen, exploits host and environmental pH cues to transition between planktonic, biofilm, and virulence-associated states. However, precise tools to monitor bacterial pH dynamics at subcellular resolution have been limited. Herein, we report the application of mCherryTYG, a genetically encoded pH-sensitive fluorophore optimized for fluorescence lifetime imaging microscopy (FLIM), enabling robust and high-resolution pH measurements across diverse conditions. mCherryTYG demonstrated exceptional sensitivity across a broad pH range (5.5-8.5) with consistent lifetime responses and was unaffected by temperature, buffer composition, or ionic strength. Using FLIM, we characterized the pH dynamics of <i>Salmonella</i> across <i>in vitro,</i> host, and biofilm contexts. Under acidic stress <i>in vitro</i>, <i>Salmonella</i> maintained a uniform intracellular pH (∼6.04), providing clarity on previously debated heterogeneity. In infections of HeLa cells, <i>Salmonella</i> existed in distinct pH environments: acidic vacuolar pH (∼5.89) and neutral pH (∼7.10). During the late infection stage, ∼17% of the bacterial population retained an acidic pH. Biofilm studies revealed stratified pH profiles with acidic pH near the bottom and neutral pH at the surface, mirroring patterns observed in other pathogens. In heterologous host models, pH gradients shape bacterial adaptation strategies. In <i>C. elegans</i>, <i>Salmonella</i> experienced a progressive internal pH gradient from neutral pH (∼7.10) in the anterior lumen to acidic pH (∼6.45) in the posterior. Similarly, in zebrafish, <i>Salmonella</i> encountered acidic lysosome-rich enterocytes (∼5.84) and neutral regions (∼7.33) in the anterior gut. This study establishes mCherryTYG-FLIM as a transformative tool for studying bacterial pH regulation, revealing pH as a critical modulator of <i>Salmonella</i> lifestyle transitions between virulence and persistence. Our findings provide new insights into host-microbe interactions and present pH as a promising target for therapeutic interventions against bacterial infections.