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

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.