Modulação de genes e de seus produtos afeta a virulência de Streptococcus mutans
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Data
2019-09-18
Autores
Castillo Pedraza, Midian Clara [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Streptococcus mutans orquestra a formação de biofilmes cariogênicos através da produção da matriz extracelular que contém exopolissacarídeos (EPS), DNA extracelular (eDNA) e ácidos lipoteicóicos (LTA). EPS são um marcador de virulência para cárie, mas não está claro como os genes associados com eDNA (lytS e lytT) e LTA (dltA e dltD) afetam a virulência de S. mutans. Portanto, avaliou-se como os genes lytST, dltAD e gtfB (EPS-insolúveis) afetariam o desenvolvimento de lesões cariosas em ratos e a sobrevivência de larvas (Galleria mellonella) após infecção sistêmica e para esclarecer sua contribuição na patogenicidade dessa bactéria. Ainda, avaliou-se o efeito dos tratamentos tópicos com miricetina (afeta a síntese de EPS), composto 1771 (modula o metabolismo do LTA) e flúor (prevenção da cárie) sobre biofilmes in vitro de S. mutans. Alimentou-se os ratos com dieta cariogênica e inoculou-se com cepa parental UA159 e cepas com deleção de genes ∆lytS, ∆dltD e ΔgtfB (n=14). Após 5 semanas, avaliou-se as populações microbianas (cultivável total e S. mutans) e as lesões de cárie. Injetou-se a cepa parental e as cepas com deleção de genes ΔgtfB, ΔlytS, ΔlytT, ΔdltA e ΔdltD na hemocele de larvas e registrou-se a sobrevivência das larvas longitudinalmente (n=10). Formou-se biofilmes de S. mutans sobre discos de hidroxiapatita revestidos com película salivar e realizou-se tratamentos tópicos duas vezes ao dia: miricetina (Mir), composto 1771, flúor (F) Mir+1771, Mir+F, 1771+F, Mir+1771+F e veículo (controle) (n=6). Determinou-se nesses biofilmes diversos parâmetros: biomassa, população, componentes da matriz (EPS solúveis e insolúveis em água, eDNA e LTA), arquitetura tridimensional e expressão gênica. Ainda, avaliou-se a citotoxicidade dos tratamentos (n=8). Na análise da população microbiana em ratos, a proporção de S. mutans na microbiota total recuperada foi maior para o grupo UA159 versus os grupos com deleção (15, 3 e 6 vezes para ΔgtfB, ∆lytS e ΔdltD, respectivamente). UA159 produziu maior número de lesões de cárie, com maior severidade (lesões em dentina): no esmalte (50% mais lesões) e na dentina (80% mais cavidades) de superfícies lisas e superfície sulcal (30% mais lesões em esmalte e 60% mais lesões em dentina) ao ser comparada com as cepas com deleção. A sobrevivência de G. mellonella foi menor em larvas infectadas com UA159, seguido de ΔgtfB quando comparado com as outras cepas com deleção gênica. Tratamentos com miricetina e composto 1771 isolados resultaram em biofilmes com menores contagens de S. mutans, biomassa, EPS solúveis e insolúveis (vs. veículo). Entretanto, a combinação Mir+1771+F foi mais eficaz, pois diminuiu a contagem de S. mutans (3 logs), biomassa (60%), EPS solúveis (74%) e insolúveis (87,5%) (vs. veículo). Ainda, essa estratégia afetou negativamente eDNA, LTA, alterou o tamanho das microcolônias e inibiu a expressão dos genes gtfBC. Adicionalmente, os tratamentos Mir+1771 e Mir+F (vs. controle de viabilidade) foram levemente citotóxicos (redução de viabilidade celular em 13% e 25%, respectivamente). Portanto, os genes lytST, dltAD e gtfB contribuem para a cariogenicidade e a virulência de S. mutans; a estratégia com miricetina e composto 1771 modula os produtos destes genes, tornando-a promissora para o controle desses biofilmes.
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.
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.
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Palavras-chave
Cárie dentária, Streptococcus mutans, Placa dentária, Dental caries, Streptococcus mutans, Dental biofilm