Quorum sensing rewires membrane vesicle protein cargo to promote antibiotic persistence in Streptococcus mutans

Quorum sensing (QS) plays a central role in the adaptive biology of Streptococcus mutans, yet the extent to which competence-stimulating peptide (CSP) signaling intersects with membrane vesicle production and function remained unclear. In this study, CSP activation markedly increased membrane vesicle output during the stationary phase and drove extensive changes in vesicle protein composition. Proteomic analysis revealed broad remodeling of vesicle cargo, including shifts in subcellular origin, and enrichment of multiple functional protein classes. Notably, CSP-induced vesicles carried the persistence-associated peptide Pep299, suggesting a link between QS-dependent cargo loading and antibiotic survival. To examine the functional consequences of this remodeling, S. mutans cells were pre-exposed to vesicles prior to antibiotic challenge. Vesicles derived from CSP-induced cultures promoted persister formation in a concentration-dependent manner, whereas vesicles from non-induced cultures exhibited minimal activity. Wild-type vesicles increased persistence, and vesicles from Pep299-overexpressing cells further enhanced this phenotype at lower vesicle concentrations. In contrast, vesicles derived from a Δ299 mutant failed to promote persistence, demonstrating that Pep299 is a key determinant of this activity. At higher vesicle abundance, the responses of wild-type and Pep299-enriched vesicles converged, consistent with endogenous CSP-dependent Pep299 loading. Fusion assays using R18 dequenching showed that vesicles from wild-type, Pep299-overexpressing, and Δ299 strains fused with comparable efficiency, indicating that differences in persistence arise from cargo composition rather than altered delivery efficiency. Together, these findings reveal that CSP QS coordinates biogenesis and selective cargo remodeling in S. mutans and identify Pep299-containing vesicles as a quorum-regulated mechanism promoting antibiotic persistence.IMPORTANCEQuorum sensing enables bacterial populations to coordinate adaptive behaviors, yet its influence on membrane vesicle biology is not fully understood. This study shows that competence-stimulating peptide (CSP) signaling in Streptococcus mutans not only increases vesicle production but also remodels vesicle protein cargo, including enrichment of the persistence-associated peptide Pep299. CSP-induced vesicles enhance the formation of persister cells through a cargo-dependent mechanism rather than through changes in vesicle-cell fusion. These findings uncover a previously unrecognized connection between quorum sensing and vesicle-mediated persistence and reveal a strategy through which S. mutans modulates community behavior and resilience within the oral biofilm environment.