Mechanosensitive channels MscL and MscS as potential targets for foodborne Pseudomonas fluorescens control with assistance of ultrasound.

Ultrasound is an emerging technology for cleaning produce and surface sterilization in the food industry; however, inappropriate intensity of ultrasound treatment may induce stress responses in food spoilage bacteria and enhance their stress resistance. This study systematically evaluated roles of mechanosensitive (MS) channel proteins: MscL and MscS on ultrasonic stress responses in Pseudomonas fluorescens as a typical bacterium leading to food spoilage. The MS channel proteins deletion mutants of P. fluorescens ATCC 13525 were initially constructed, including ΔmscL, ΔmscS, and ΔmscL ΔmscS. MscL was more sensitive to mechanical stimulation of ultrasound which rapidly released intracellular osmotic pressure to alleviate membrane tension damage; however, MscS only provided complementary protection under the ultrasound treatment as proved by Ca2+ fluorescence labeling and antibacterial activity assays. Acoustic pressure distribution and thresholds of MscL (289.45 kPa) and MscS (334.21 kPa) initiating acoustic stress response were determined by COMSOL Multiphysics. Phenotypic assays showed that ultrasound induced membrane potential disruption, ATP depletion, leakage of intracellular components, and oxidative stress response in an intensity-dependent manner with the strongest disturbance observed in the ΔmscL ΔmscS mutant. Ten stress-related genes expression at different ultrasonic intensities further indicated that MscL primarily governed the initial response to ultrasound stimulation, whereas MscS acted as a regulatory factor during sustained and amplified signaling. Both MscL and MscS maintained membrane integrity and cellular homeostasis which jointly regulated the initial transcriptional response including oxidative stress, biofilm formation, and DNA repair. Results of this study would provide theoretical evidences for ultrasound-activation of MS proteins to achieve efficient antibacterial activity.