FLIM of mCherryTYG Deciphers pH Dynamics and Lifestyles of Salmonella Typhimurium

Intracellular pH regulation is fundamental to bacterial
adaptation,
virulence, and survival in diverse environments. Salmonella 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 Salmonella across in vitro, host, and biofilm contexts. Under acidic stress in vitro, Salmonella maintained a uniform intracellular
pH (∼6.04), providing clarity on previously debated heterogeneity.
In infections of HeLa cells, Salmonella 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 C. elegans, Salmonella 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, Salmonella 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 Salmonella 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.