Celine Marie Levesque

Volume 208, no. 8, e00323-26, 2026, https://doi.org/10.1128/jb.00323-26. The Acknowledgments section was inadvertently omitted from the published version of the article. The section should read as follows: “The authors gratefully acknowledge the University of Toronto Centre for the Analysis of Genome Evolution and Function (CAGEF) for proteomics support and sincerely thank Dr. Pauline Wang and Inga Kireeva for their expertise and assistance with proteomic analyses. The authors also thank Dr. Zhao for helpful discussions and valuable scientific input.

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. IMPORTANCE: Quorum 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.

Streptococcus mutans , a key oral pathogen, utilizes quorum sensing to regulate biofilm formation—a major virulence factor in the development of dental caries (tooth decay). Our recent research uncovered a complex interplay between the CSP-ComDE quorum sensing pathway and the Type II DpnII restriction-modification (R-M) system in S. mutans . The DpnII R-M system methylates adenine at 5′-GATC-3′ sites and cleaves unmethylated DNA, significantly influencing foreign DNA acquisition and gene expression. In this study, we investigated the impact of a ΔRM mutant, which lacks adenine methylation, on biofilm formation. The ΔRM mutant formed fragile biofilms that easily detach from surfaces, with significantly reduced exopolysaccharide content and increased extracellular DNA, which appears to be associated with membrane vesicle production rather than cell lysis. RNA-seq analysis revealed only few differentially expressed genes directly involved in biofilm formation, such as gtfC , suggesting that the biofilm defect may result from indirect effects or alternative regulatory mechanisms. Notably, the downregulation of mutanobactin-related genes and upregulation of genes involved in de novo purine nucleotide biosynthesis point to novel pathways influenced by DNA methylation. These findings contribute to a deeper understanding of the multifactorial nature of biofilm formation and the role of epigenetic modifications in microbial behavior. IMPORTANCE: This study highlights the critical role of DNA methylation in regulating biofilm formation and virulence in Streptococcus mutans . By examining the interplay between adenine methylation, extracellular DNA (eDNA), membrane vesicles (MVs), and glucan production, we provide new insights into the complex biology of biofilm development. Our findings challenge traditional views by emphasizing the importance of MVs and eDNA in maintaining biofilm integrity. Understanding these epigenetics modifications not only advances our knowledge of microbial regulation but also identifies novel targets for antimicrobial therapy. Since adenine methylation is rare or absent in mammalian cells, targeting this modification presents a promising strategy to disrupt biofilm formation and combat bacterial infections. The insights gained from this study may inform the development of innovative approaches to manage biofilm-associated infections and improve oral health outcomes.

Streptococcus mutans, a key player in dental caries, faces multiple environmental challenges within the oral cavity, including oxidative stress, nutrient scarcity, and acidic pH. To survive and thrive, S. mutans has evolved intricate mechanisms, including the CSP‐ComDE quorum sensing system, which coordinates responses to environmental cues. The CSP‐ComDE system enables S. mutans to communicate with neighboring cells via its CSP pheromone. Under stress conditions, the CSP pheromone production increases, triggering a cascade of events. Notably, our research demonstrated that the CSP pheromone activates the expression of a Type II restriction‐modification (R‐M) system. Type II R‐M systems are well‐known tools in molecular biology and genetic engineering and consist of two distinct enzymes: a restriction enzyme and a methyltransferase. An increasing number of studies have revealed that bacterial adenine methylation (Dam methylation) has a broader role beyond mere DNA protection. In fact, the marks introduced into the DNA provide signals for a variety of physiological processes. Our results highlight a conserved chromosomal locus in S. mutans encoding the DpnII R‐M system. DpnII R‐M methylates DNA at 5′‐GATC target sites within the S. mutans genome and cleaves unmarked DNA. Furthermore, our findings suggest that Dam methylation significantly impacts foreign DNA acquisition via natural transformation and modulates mutanobactin expression—a secondary metabolite linked to oxidative stress tolerance. Collectively, our findings suggest that Dam methylation bridges epigenetics and bacterial fitness, potentially opening new avenues in bacterial epigenetics. As we explore this intricate biological process, we may uncover novel therapeutic strategies to combat bacterial infections.

Persisters are a small fraction of growth-arrested phenotypic variants that can survive lethal concentrations of antibiotics but are able to resume growth once antibiotics are stopped. Their formation can be a stochastic process or one triggered by environmental cues. In the human pathogen Streptococcus mutans, the canonical peptide-based quorum-sensing system is an inducible DNA repair system that is pivotal for bacterial survival. Previous work has shown that the CSP-signaling peptide is a stress-signaling alarmone that promotes the formation of stress-induced persisters. In this study, we exposed S. mutans to the CSP pheromone to mimic DNA damage conditions and isolated the antibiotic persisters by treating the cultures with ofloxacin. A transcriptome analysis was then performed to evaluate the differential gene expression between the normal stationary-phase cells and the persisters. RNA sequencing revealed that triggered persistence was associated with the upregulation of genes related to several stress defense mechanisms, notably, multidrug efflux pumps, the arginine deaminase pathway, and the Opu/Opc system. In addition, we showed that inactivation of the VicK kinase of the YycFG essential two-component regulatory system abolished the formation of triggered persisters via the CSP pheromone. These data contribute to the understanding of the triggered persistence phenotype and may suggest new therapeutic strategies for treating persistent streptococcal infections.

Bacteria use quorum sensing (QS) to communicate with each other via secreted small autoinducers produced by individuals. QS allows bacteria to display a unified response that benefits the species during adaptation to environment, colonization, and defense against competitors. In oral streptococci, the CSP-ComDE QS is an inducible DNA damage repair system that is pivotal for bacterial survival. In the oral pathogen Streptococcus mutans, the QS system positively influences the formation of antibiotic persisters, cells that can survive antibiotic attack by entering a non-proliferative state. We recently identified a novel gene, pep299, that is activated in the persister cell fraction induced by QS. In this study, we focused our investigation on the role of pep299, a gene encoding a bacteriocin-like peptide, in the formation of antibiotic persisters. Mutant Δ299, unable to produce Pep299, showed a dramatic reduction in the number of stress-induced persisters. Using a co-culture assay, we showed that cells overproducing pep299 induced the formation of persisters in the mutant, suggesting that Pep299 was actively secreted and detected by neighboring cells. Cells exposed to DNA damage conditions activated the gene expression of pep299. Interestingly, our results suggested that the pep299 gene was also involved in the regulation of a QS-inducible toxin–antitoxin system. Our study suggests that the pep299 gene is at the core of the triggered persistence phenotype in S. mutans, allowing cells to transition into a state of reduced metabolic activity and antibiotic tolerance.

Biomaterial–dentin interfaces undergo degradation over time, allowing salivary, tissue fluid, and bacterial movement between the root filling or restoration and dentin. This study aims to investigate the effect of aging in simulated human salivary/bacterial/blood esterases (SHSE) on proliferation and viability of Enterococcus faecalis biofilm within the dentin interface with four materials used to fill/restore the endodontic space. Root canals of human anterior teeth were prepared and filled with gutta-percha and one of the following: self-cured resin composite (Bisfil^TM 2B, Bisco, Schaumburg, IL, USA) with either self-etch (SE) (EasyBond) or total-etch (TE) (Scotchbond^TM, 3M, Saint Paul, MN, USA) methacrylate-based adhesives, epoxy-resin sealer (AH Plus^®, Dentsply Sirona, York, PA, USA), or bioceramic sealer (EndoSequence^® BC Sealer™, Brasseler USA, Savannah, GA, USA). Specimens were aged in SHSE or phosphate-buffered saline (PBS) for up to 360 days, followed by cultivation of steady-state E. faecalis biofilm. Depth and viability of interfacial bacterial biofilm proliferation were assessed by confocal laser scanning microscopy and live/dead staining. Data were analyzed using three-way ANOVA and Scheffe’s post hoc analyses. Initial depths of biofilm proliferation were similar among material groups (p > 0.05). All groups showed significantly deeper biofilm proliferation with increased aging period (p < 0.05). SHSE aging increased interfacial biofilm depth for TE, SE and BC (p < 0.05) but not AH. For unaged interfaces, BC exhibited the lowest ratio of live bacteria, followed by AH, TE, and SE (p < 0.05). Interfacial bacterial biofilm proliferation and viability were dependent on the biomaterial, aging media, and period.

Enterococcus faecalis as an important nosocomial pathogen is critically implicated in the pathogenesis of endocarditis, urinary tract, and persistent root canal infections. Its major virulence attributes (biofilm formation, production of proteases, and hemolytic toxins) enable it to cause extensive host tissue damage. With the alarming increase in enterococcal resistance to antibiotics, novel therapeutics are required to inhibit E. faecalis biofilm formation and virulence. Trans-cinnamaldehyde (TC), the main phytochemical in cinnamon essential oils, has demonstrated promising activity against a wide range of pathogens. Here, we comprehensively investigated the effect of TC on planktonic growth, biofilm formation, proteolytic and hemolytic activities, as well as gene regulation in E. faecalis. Our findings revealed that sub-inhibitory concentrations of TC reduced biofilm formation, biofilm exopolysaccharides, as well as its proteolytic and hemolytic activities. Mechanistic studies revealed significant downregulation of the quorum sensing fsr locus and downstream gelE, which are major virulence regulators in E. faecalis. Taken together, our study highlights the potential of TC to inhibit E. faecalis biofilm formation and its virulence.

Enterococcus faecalis as an important nosocomial pathogen is critically implicated in the pathogenesis of endocarditis, urinary tract and surgical wound infections. Its major virulence attributes (biofilm formation, production of proteases and hemolytic toxins) enable it to cause extensive host tissue damage. With the alarming increase in enterococcal resistance to antibiotics, novel therapeutics are required to inhibit E. faecalis biofilm formation and virulence. Trans -cinnamaldehyde (TC), the main phytochemical in cinnamon essential oils has demonstrated promising activity against a wide range of pathogens. Here, we comprehensively investigated the effect of TC on planktonic growth, biofilm formation, proteolytic and hemolytic activities, as well as gene regulation in E. faecalis . Our findings revealed that sub-inhibitory concentrations of TC reduced biofilm formation, biofilm exopolysaccharides as well as its proteolytic and hemolytic activities. Mechanistic studies revealed significant down regulation of the quorum sensing fsr locus and downstream gelE , which are major virulence regulators in E. faecalis . Taken together, our study highlights the potential of TC to inhibit E. faecalis biofilm formation and its virulence.