Ultrasound cavitation modulates intracellular ROS and gene expression in antibacterial sonodynamic therapy

• Ultrasound stimulation induces mild endogenous ROS production in bacterial cells, as quantified by DCFH-DA and flow cytometry. • Antibacterial efficacy shows dose-dependence on ultrasound cavitation intensity. • The primary mechanism of aSDT involves cavitation-induced mechanical damage to bacterial membranes and surface structures, coupled with US/CUR-synergized extracellular ROS generation. • The synergistic effect of ultrasound cavitation and sonosensitizers significantly enhances antibacterial efficacy through two key mechanisms. • Markedly downregulating PA4211, PA0876, and PA3361 expression to disrupt biofilm structure and inhibit its remodeling, ultimately causing irreversible membrane damage. • Substantially upregulating PA3570, PA2433, and PA4880 expression to disturb redox homeostasis and metabolic balance, leading to massive intracellular ROS accumulation. Antibacterial sonodynamic therapy (aSDT) has emerged as a promising non-invasive antimicrobial modality utilizing ultrasound activation. This study employed curcumin (CUR) as a sonosensitizer to systematically investigate the mechanisms underlying aSDT. We first examined ultrasound (US)-induced intracellular reactive oxygen species (ROS) generation and subsequently evaluated the bactericidal enhancement mediated by microbubble-augmented cavitation, including membrane disruption and apoptotic pathways. A comprehensive experimental approach was implemented, including: (1) bacterial viability assays on solid media coupled with biomass quantification, (2) flow cytometric apoptosis detection, (3) intracellular ROS measurement, (4) gene expression changes through RNA-seq and qRT-PCR, and (5) scanning electron microscopy (SEM) analysis of Pseudomonas aeruginosa morphology. Results demonstrated a dose-dependent relationship between cavitation intensity and antibacterial efficacy. Although US can stimulate intracellular ROS elevation, experimental results indicated that aSDT operates primarily through two mechanisms: ultrasound-induced mechanical disruption of cell membranes, and apoptosis triggered by extracellular ROS generated via US/sonosensitizer synergy that enter cells. Genomic analysis further elucidated that aSDT-induced bacterial apoptosis originated from the synergistic effect of ultrasonic cavitation and sonosensitizer. It differentially regulated the expression of key genes in P. aeruginosa (downregulating PA4211, PA0876, PA3361 and upregulating PA3570, PA2433, PA4880), damaged the biofilm structure and inhibited its remodeling. Meanwhile, it disrupted the redox homeostasis and metabolic balance, utimately leading to irreversible damage to the cell membrane and a massive accumulation of intracellular ROS.