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| Author(s): |
Midian Clara Castillo Pedraza
Total Authors: 1
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| Document type: | Doctoral Thesis |
| Press: | Araraquara. 2019-09-24. |
| Institution: | Universidade Estadual Paulista (Unesp). Faculdade de Odontologia. Araraquara |
| Defense date: | 2019-09-18 |
| Advisor: | Marlise Inêz Klein; Pedro Luiz Rosalen |
| Abstract | |
Streptococcus mutans orchestrates the cariogenic biofilms formation through the production of an extracellular matrix that contains exopolysaccharides (EPS), extracellular DNA (eDNA), and lipoteichoic acids (LTA). EPS is a virulence marker of dental caries, but it is unclear how genes associated with eDNA (lytS and lytT) and LTA (dltA and dltD) affect S. mutans virulence. Therefore, this study evaluated how the genes lytST, dltAD and gtfB (insoluble EPS) affected the development of carious lesions in rats and the survival of larvae (Galleria mellonella) after systemic infection to clarify its contribution to the pathogenicity of S. mutans. Also, it assessed the effect of topical treatments with myricetin (affects EPS synthesis), compound 1771 (modulates LTA metabolism) and fluoride (caries prevention) on in vitro S. mutans biofilms. The rats received a cariogenic diet and were inoculated with the parental strain UA159 and its strains with deletion of genes ΔlytS, ΔdltD, and ΔgtfB (n=14). After five weeks, viable microbial populations (total cultivable and S. mutans) and caries lesions were evaluated. Larvae were inoculated by intra-hemocoel injection with the parental strain and deletion strains ΔgtfB, ΔlytS, ΔlytT, ΔdltA, and ΔdltD, and larval survival was recorded longitudinally (n=10). S. mutans in vitro biofilms were formed on saliva-coated hydroxyapatite discs and topically treated twice-daily: Myricetin (Myr), Compound 1771, Fluoride (F), Myr+1771, Myr+F, 1771+F, Myr+1771+F, and vehiclecontrol (n=6). The biofilms were evaluated to determine biomass, viable S. mutans counts, matrix components (water-soluble and -insoluble EPS, eDNA, and LTA), tridimensional architecture, and gene expression. Also, the cytotoxicity of the treatments was evaluated. In the rodent caries model, the proportion of S. mutans in total microbiota recovered was higher for the UA159 group versus the deletion groups (15, 3 and 6-fold for ΔgtfB, ΔlytS, and ΔdltD, respectively). The parental strain UA159 yielded higher number of caries lesions that were also more severe (lesions in dentin) : on enamel (50% more lesions) and dentin (80% more cavities) of smooth surfaces, and on enamel (30% more lesions) and dentin (60% more lesions) sulcal surfaces when compared to the deletion strains. The G. mellonella survival was lower in UA159- infected larvae, followed by ΔgtfB-infected when compared to other gene deletion strains. Topical treatments with either myricetin or compound 1771 alone led the biofilms with lower amounts of S. mutans viable counts, biomass, water-soluble, and water-insoluble EPS (vs. vehicle). However, the combination Myr+1771+F was more efficient, as it decreased 3 logs of S. mutans counts, 60% of the biomass, 74% of % water-soluble EPS, and 87.5% of water-insoluble EPS counts (vs. vehicle). Also, this strategy negatively affected eDNA, LTA, altered the size of microcolonies, and inhibited the expression of gtfBC genes. Additionally, Mir+1771 and Mir+F (25%) treatments (vs. viability control) were mildly cytotoxic (reduction of viability by 13% and 25%, respectively). Therefore, the genes lytST, dltAD, and gtfB contribute to the cariogenicity and virulence of S. mutans; a strategy with myricetin and compound 1771 modulates the products of these genes, making it a promissory approach to control cariogenic biofilms. (AU) | |
| FAPESP's process: | 17/00753-0 - In vivo virulence analyses of Streptococcus mutans lytTS genes and operon dltABCD and modulation thereof by targeted therapy |
| Grantee: | Midian Clara Castillo Pedraza |
| Support Opportunities: | Scholarships in Brazil - Doctorate |