Função de DNA extracelular e de ácido lipoteicóico nas propriedades estruturais e funcionais da matriz extracelular de biofilmes cariogênicos
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Data
2016-08-12
Autores
Castillo Pedraza, Midian Clara [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
A cárie dentária é uma doença dependente de biofilme, da dieta e fatores de hospedeiro. O biofilme é uma comunidade microbiana dinâmica, coberta pela matriz extracelular (MEC). Em biofilmes cariogênicos, a MEC rica em exopolissacarídeos proporciona microambientes ácidos, causando desmineralização do esmalte. Este estudo avaliou a função de DNA extracelular (eDNA) e ácido lipoteicóico (ALT) nas propriedades estruturais e funcionais da MEC de biofilmes cariogênicos. Biofilmes monoespécie de Streptococcus mutans UA159 (cepa parental ou mutantes knockout) e mistos (UA159 cepa parental ou mutantes, Actinomyces naeslundii ATCC 12104 e Streptococcus gordonii DL-1) foram formados em discos de hidroxiapatita com película salivar, em meio com saliva e 0,1% sacarose, alternado com 0,5% sacarose+1% amido (37°C/5% CO2). O pH do meio de cultura foi verificado a cada troca. Para modular componentes da MEC foram utilizadas cepas mutantes de S. mutans para os genes lytTS (∆SMu.525 e ∆SMu.526 - eDNA), operon dltABCD (∆SMu.1538 e ∆SMu.1541 – ALT) e gtfB (exopolissacarídeos insolúveis). A população microbiana foi avaliada longitudinalmente. Nas idades do biofilme 67 e 115 h, os biofilmes foram processados para avaliar a biomassa, as proteínas totais, e os componentes da matriz: eDNA, ALT, proteínas e exopolissacarídeos solúveis (WSP) e insolúveis (ASP) em água. Os dados foram analisados com ANOVA um critério e teste de Tukey (α=0,05). Na presença de 0,5% sacarose+1% amido, o pH do meio tornou-se mais ácido. Nos estágios iniciais dos biofilmes mistos, S. gordonii foi predominante (p<0,05); a proporção das três espécies foi similar em 67 h, e com o passar do tempo S. mutans se tornou majoritário (p<0,05), independentemente da modulação dos componentes da MEC. A biomassa e as proteínas totais dos biofilmes monoespécie e mistos, geralmente aumentaram significativamente com o tempo (p<0,05). Porém, detectou-se diferentes quantidades de componentes da matriz, dependendo do gene inativado e do tipo de biofilme. As cepas ∆SMu.525 e ∆SMu.526 apresentaram as maiores quantidades de eDNA na MEC em biofilme monoespécie nas duas fases de formação (p<0,05), mas no biofilme misto apenas na idade 115 h (p<0,05). Portanto, os genes lytTS modulam eDNA na MEC de biofilmes cariogênicos. ALT foi detectado na MEC de todos os biofilmes, ocorrendo aumento significativo com a idade em biofilmes monoespécie das cepas ∆SMu.1538 e ∆SMu.1541 (vs. UA159; p<0,05). Houve variação na quantidade de WSP e ASP na MEC dos biofilmes monoespécie e mistos. Em biofilmes com a cepa gtfB existe menos WSP nas duas idades em monoespécie (p<0,05), e em mistos não houve variação (p>0.05). Ocorre menos WSP na MEC de biofilmes monoespécie de ΔSMu.525 na idade 115 h (vs. UA159). A quantidade de ASP no biofilme misto com 115 h foi menor para a cepa gtfB em relação aos genes do operon dltABCD (p<0,05). Finalmente, o conteúdo de proteínas na MEC foi menor para a cepa gtfB versus UA159 e ∆SMu.1541 em biofilme misto de 115 h (p<0,05). Portanto, eDNA, ALT e exopolissacarídeos atuando em conjunto, contribuiram para a aderência de microrganimos, formação e maturação de biofilmes cariogênicos.
Dental caries is a disease dependent of biofilm, diet and host factors. Biofilm is a dynamic microbial community enmeshed by an extracellular matrix (ECM). In cariogenic biofilms, the ECM rich in exopolysacharides provide acidic microenvironments that cause demineralization of enamel. The present study evaluated the role of extracellular DNA (eDNA) and lipoteichoic acid (LTA) in the structural and functional properties of the matrix of cariogenic biofilm. Single-species biofilms of Streptococcus mutans UA159 (parental or knockout strains) and mixed-species (UA159 parental or mutant strains, Actinomyces naeslundii ATCC 12104 and Streptococcus gordonii DL-1) were grown onto saliva-coated hydroxyapatite discs, in medium with saliva and 0.1% sucrose, alternating with 0.5% sucrose + 1% starch (37°C / 5%CO2). The pH of spent media was verified at each medium change. The S. mutans knockout mutant strains of genes lytTS (ΔSMu.525 and ΔSMu.526 - eDNA), operon dltABCD (ΔSMu.1538 and ΔSMu.1541 - LTA) and gtfB (insoluble exopolysaccharide) were used to modulate ECM´ components. The microbial population was evaluated longitudinaly. At 67 and 115 h, biofilms were processed to assess biomass, total protein and ECM components: eDNA, LTA, soluble proteins, and water soluble (WSP) and insoluble (ASP) exopolysaccharides. The data were analyzed by ANOVA one-way and Tukey test (α=0.05). In the presence of 0.5% sucrose + 1% starch the pH of spent media became more acid. During the initial stages of mixed-species biofilm development, S. gordonii prevail (p<0.05); the proportion of the three species was similar at 67 h, and S. mutans becomes the dominant species over time (p<0.05), independently of ECM´ components modulation. The biomass and the total protein content of single- and mixed-species biofilms usually showed significant increases over time (p<0.05). However, distinct amounts of EMC´s components were detected, depending on the gene that was inactivated and on the biofilm type. The strains ΔSMu.525 and ΔSMu.526 showed the highest amounts of eDNA in ECM of single-species biofilms in both ages (p<0.05), but in mixed-species biofilms only at 115 h (p<0.05). Therefore, the genes lytTS modulate eDNA in the ECM of cariogenic biofilms. LTA was detected in the matrix of all biofilms, significantly increasing as the single-species biofilms of ∆SMu.1538 and ∆SMu.1541 aged (vs. UA159; p<0.05). The amount of WSP and ASP varied in both biofilm types. The strain gtfB formed single-species biofilms with less WSP in the ECM at 67 and 115 h (p<0.05), but in mixed-species biofilms no significant change occured (p>0.05). There is less WPS in the matrix of single-species biofilms formed by mutant ΔSMu.525 at 115 h (vs. UA159). The amount of ASP in mixed-species biofilm with age 115 h was lower for gtfB strain compared to dltABCD operon genes (p<0.05). Finally, the amount of proteins in the ECM was lower only for strain gtfB versus UA159 and ΔSMu.1541 in mixed-species biofilm at 115 h (p<0.05). Therefore, eDNA, ALT and ESP working together contribute to the microbial adhesion, formation and maturation of cariogenic biofilms.
Dental caries is a disease dependent of biofilm, diet and host factors. Biofilm is a dynamic microbial community enmeshed by an extracellular matrix (ECM). In cariogenic biofilms, the ECM rich in exopolysacharides provide acidic microenvironments that cause demineralization of enamel. The present study evaluated the role of extracellular DNA (eDNA) and lipoteichoic acid (LTA) in the structural and functional properties of the matrix of cariogenic biofilm. Single-species biofilms of Streptococcus mutans UA159 (parental or knockout strains) and mixed-species (UA159 parental or mutant strains, Actinomyces naeslundii ATCC 12104 and Streptococcus gordonii DL-1) were grown onto saliva-coated hydroxyapatite discs, in medium with saliva and 0.1% sucrose, alternating with 0.5% sucrose + 1% starch (37°C / 5%CO2). The pH of spent media was verified at each medium change. The S. mutans knockout mutant strains of genes lytTS (ΔSMu.525 and ΔSMu.526 - eDNA), operon dltABCD (ΔSMu.1538 and ΔSMu.1541 - LTA) and gtfB (insoluble exopolysaccharide) were used to modulate ECM´ components. The microbial population was evaluated longitudinaly. At 67 and 115 h, biofilms were processed to assess biomass, total protein and ECM components: eDNA, LTA, soluble proteins, and water soluble (WSP) and insoluble (ASP) exopolysaccharides. The data were analyzed by ANOVA one-way and Tukey test (α=0.05). In the presence of 0.5% sucrose + 1% starch the pH of spent media became more acid. During the initial stages of mixed-species biofilm development, S. gordonii prevail (p<0.05); the proportion of the three species was similar at 67 h, and S. mutans becomes the dominant species over time (p<0.05), independently of ECM´ components modulation. The biomass and the total protein content of single- and mixed-species biofilms usually showed significant increases over time (p<0.05). However, distinct amounts of EMC´s components were detected, depending on the gene that was inactivated and on the biofilm type. The strains ΔSMu.525 and ΔSMu.526 showed the highest amounts of eDNA in ECM of single-species biofilms in both ages (p<0.05), but in mixed-species biofilms only at 115 h (p<0.05). Therefore, the genes lytTS modulate eDNA in the ECM of cariogenic biofilms. LTA was detected in the matrix of all biofilms, significantly increasing as the single-species biofilms of ∆SMu.1538 and ∆SMu.1541 aged (vs. UA159; p<0.05). The amount of WSP and ASP varied in both biofilm types. The strain gtfB formed single-species biofilms with less WSP in the ECM at 67 and 115 h (p<0.05), but in mixed-species biofilms no significant change occured (p>0.05). There is less WPS in the matrix of single-species biofilms formed by mutant ΔSMu.525 at 115 h (vs. UA159). The amount of ASP in mixed-species biofilm with age 115 h was lower for gtfB strain compared to dltABCD operon genes (p<0.05). Finally, the amount of proteins in the ECM was lower only for strain gtfB versus UA159 and ΔSMu.1541 in mixed-species biofilm at 115 h (p<0.05). Therefore, eDNA, ALT and ESP working together contribute to the microbial adhesion, formation and maturation of cariogenic biofilms.
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Palavras-chave
Cárie dentária, Placa dentária, Streptococcus mutans, Dental caries, Dental biofilm