The copYAZ Operon Functions in Copper Efflux, Biofilm Formation, Genetic Transformation, and Stress Tolerance in Streptococcus mutans.

UNLABELLED: In bacteria, copper homeostasis is closely monitored to ensure proper cellular functions while avoiding cell damage. Most Gram-positive bacteria utilize the copYABZ operon for copper homeostasis, where copA and copB encode copper-transporting P-type ATPases, whereas copY and copZ regulate the expression of the cop operon. Streptococcus mutans is a biofilm-forming oral pathogen that harbors a putative copper-transporting copYAZ operon. Here, we characterized the role of copYAZ operon in the physiology of S. mutans and delineated the mechanisms of copper-induced toxicity in this bacterium. We observed that copper induced toxicity in S. mutans cells by generating oxidative stress and disrupting their membrane potential. Deletion of the copYAZ operon in S. mutans strain UA159 resulted in reduced cell viability under copper, acid, and oxidative stress relative to the viability of the wild type under these conditions. Furthermore, the ability of S. mutans to form biofilms and develop genetic competence was impaired under copper stress. Briefly, copper stress significantly reduced cell adherence and total biofilm biomass, concomitantly repressing the transcription of the gtfB, gtfC, gtfD, gbpB, and gbpC genes, whose products have roles in maintaining the structural and/or functional integrity of the S. mutans biofilm. Furthermore, supplementation with copper or loss of copYAZ resulted in significant reductions in transformability and in the transcription of competence-associated genes. Copper transport assays revealed that the ΔcopYAZ strain accrued significantly large amounts of intracellular copper compared with the amount of copper accumulation in the wild-type strain, thereby demonstrating a role for CopYAZ in the copper efflux of S. mutans. The complementation of the CopYAZ system restored copper expulsion, membrane potential, and stress tolerance in the copYAZ-null mutant. Taking these results collectively, we have established the function of the S. mutans CopYAZ system in copper export and have further expanded knowledge on the importance of copper homeostasis and the CopYAZ system in modulating streptococcal physiology, including stress tolerance, membrane potential, genetic competence, and biofilm formation. IMPORTANCE: S. mutans is best known for its role in the initiation and progression of human dental caries, one of the most common chronic diseases worldwide. S. mutans is also implicated in bacterial endocarditis, a life-threatening inflammation of the heart valve. The core virulence factors of S. mutans include its ability to produce and sustain acidic conditions and to form a polysaccharide-encased biofilm that provides protection against environmental insults. Here, we demonstrate that the addition of copper and/or deletion of copYAZ (the copper homeostasis system) have serious implications in modulating biofilm formation, stress tolerance, and genetic transformation in S. mutans. Manipulating the pathways affected by copper and the copYAZ system may help to develop potential therapeutics to prevent S. mutans infection in and beyond the oral cavity.

Assessing Macrophage Polarization in Nanoparticle-Guided Wound Repair Using a Lipopolysaccharide Contaminated Intraosseous Model.

INTRODUCTION: Macrophages regulate the processes of inflammation and tissue regeneration/repair through their plasticity and phenotypes of different activation states. Previous studies have shown that disinfection of lipopolysaccharide (LPS)-contaminated dentin with photoactivated rose bengal-functionalized chitosan nanoparticles (CSRBnps) in vivo supported neotissue formation without signs of inflammation and root resorption. The aim of this study was to understand the mechanism underlying CSRBnp-guided attenuation of inflammation in LPS-contaminated dentin using macrophage polarization as an indicator of inflammation and repair. METHODS: To quantify the polarized macrophage populations, M1/M2-specific surface markers CD68, CD80, and CD206 and transcriptional factors signal transducer and activator of transcription (STAT) 1, STAT3, and STAT6 were determined using immunohistochemistry among previously obtained root specimens implanted into mandibles of guinea pigs for 4 weeks. In group 1, the canals were not inoculated; in group 2, the canals were inoculated with Pseudomonas aeruginosa LPS; in group 3, the canals were inoculated and disinfected with sodium hypochlorite; in group 4, the canals were inoculated and disinfected with sodium hypochlorite and calcium hydroxide; and in group 5, the canals were inoculated and disinfected with sodium hypochlorite, and CSRBnps (300 µg/mL) with photoactivation (λ = 540 nm, 40 J/cm2) were analyzed. RESULTS: An increased expression of M2-specific markers was observed in the group treated with CSRBnps compared with the groups treated with either conventional or no root canal disinfection. A statistically significant population of macrophages expressing both M1- and M2-specific markers was observed in all the tested groups. CONCLUSIONS: Disinfection of LPS-contaminated dentin with CSRBnps demonstrated M2-type polarization of macrophages, which corresponded to repair and neotissue formation.

Effect of two desensitizing agents on dentin hypersensitivity: A randomized split-mouth clinical trial.

BACKGROUND: Clinical research is important to evaluate the effect of desensitizing agents. AIMS: This randomized clinical trial evaluated the immediate and 1 week desensitizing effect of two desensitizing agents Uno Topical Gel and Profluorid. MATERIALS AND METHODS: Thirtyfive patients with teeth presenting with dentin hypersensitivity were included in this clinical trial. Each quadrant in a patient was randomly assigned to one of two groups: Uno Topical Gel or Profluorid Varnish. A VAS score was used to assess tooth sensitivity at baseline, immediately after application of desensitizer and after 1 week. Additionally, 30 dentin discs were prepared, divided into Group 1(Control Group), Group 2 (Profluorid Varnish) and Group 3 (Uno Topical Gel) and examined using scanning electron microscopy (SEM) after 1hour and 24 hours to evaluate tubule occlusion. STATISTICAL ANALYSIS: Clinical data were analysed using Friedman's test and Mann - Whitney U test. SEM data was analysed using Student's 2-sample t-test. RESULTS: Uno group was significantly better to evaporative stimuli immediately (P=0.01) after application. After 1 week, Uno group was significantly better to tactile (P=0.000) and evaporative (P=0.000) stimuli than Profluorid. SEM images showed that 1 hour after application, Uno and Profluorid demonstrated more than 90% and 80% dentin tubule occlusion respectively. At 24 hours, Uno and Profluorid demonstrated more than 50% and 60% dentin tubule occlusion respectively. CONCLUSIONS: Uno Topical Gel was significantly better than Profluorid in reducing pain of dentin hypersensitivity due to tactile and evaporative stimuli after 1 week.