Influência da matriz extracelular na expressão gênica de Streptococcus mutans em biofilme cariogênico
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Data
2017-05-18
Autores
Salamanca, Elkin Jahir Florez [UNESP]
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Editor
Universidade Estadual Paulista (Unesp)
Resumo
A cárie representa a doença humana mais prevalente no mundo, sua etiologia é dependente de biofilme e da dieta. Os biofilmes são comunidades altamente dinâmicas e estruturadas de células microbianas que se encontram embebidas em uma matriz extracelular tridimensional (MEC). Esta estrutura age como uma barreira que limita a difusão e fornece estabilidade e proteção aos microrganismos. Streptococcus mutans tem um potencial acidogênico e acidúrico, orquestra a construção do biofilme e modula sua virulência, produzindo ácidos lipoteicóicos (LTA), DNA extracelular (eDNA) e exopolissacarídeos (EPS), promovendo assim a adesão e coesão microbiana. Ao mesmo tempo a MEC produzida dificulta a difusão de metabólitos no biofilme. O objetivo do estudo foi determinar por meio de RT-qPCR a dinâmica da expressão de genes de S. mutans associados ao metabolismo de LTA (dltABCD, SMU_775c), eDNA (lytST, lrgAB, ccpA) e exopolissacarídeos (gtfBCD, gbpB, dexA), durante o desenvolvimento da MEC de biofilmes mistos em um estudo longitudinal. Biofilmes mistos de S. mutans UA159 (cepa parental) ou ΔgtfB (mutante com deleção do gene gtfB), Actinomyces naeslundii ATCC 12104 e Streptococcus gordonii DL-1 foram formados em discos de hidroxiapatita revestidos de película salivar, e cultivados a 37° C e 5% de CO2 em caldo triptona e extrato de levedura contendo 25% de saliva e alternando 0,1% de sacarose (escassez) e 0,5% de sacarose + 1% de amido (abundância). O pH do meio de cultura manteve-se ácido durante o período experimental, sendo menor após longos períodos de incubação. Todos os genes foram expressos em todas as idades (29, 43, 55, 67, 79, 91, 103 e 115 horas) nos dois tipos de biofilme, apresentado níveis distintos. Os genes associados ao LTA e EPS foram expressos de um modo semelhante, tendo maiores níveis de expressão duas horas após o fornecimento de carboidratos o mesmo comportamento foi registrado para as duas cepas, com maiores níveis de expressão para UA159 parental. Os genes associados a eDNA apresentaram uma dinâmica de expressão diferente entre a cepa parental e a cepa ΔgtfB. Para UA159 os genes lrgAB foram altamente expressos em 29h, e não demostram ter uma relação com os genes lytST, enquanto que no biofilme ΔgtfB observou-se uma relação inversa entre lytS e lrgAB nos horários em que o meio estava “fresco” (duas horas após a troca). Para os dois biofilmes o gene lytT apresentou os níveis de expressão mais baixos sem ter diferenças entre os horários. O produto do gene ccpA ativa ou reprime a expressão de determinados genes como resposta à disponibilidade de carboidratos, e esta relação com carboidratos foi evidente para UA159. Portanto, a deleção de gtfB influência a dinâmica de expressão dos genes avaliados, diminuindo a magnitude de expressão principalmente dos genes associados com EPS, e alternando o perfil de expressão de genes associados com a presença de eDNA na matriz extracelular.
Dental caries represents the most prevalent human disease worldwide, its etiology is biofilm-diet dependent. Biofilms are highly dynamic and structured communities of microbial cells that are enmeshed in a three-dimensional extracellular matrix (ECM). This structure acts as a diffusion-limiting barrier providing stability and protection to the microorganisms. Streptococcus mutans is acidogenic, aciduric, and orchestrates the biofilm build-up process. It modulates the biofilm’s virulence by producing lipoteichoic acids (LTA), extracellular DNA (eDNA) and exopolysaccharides (EPS), thereby promoting microbial adhesion and cohesion. The resulting ECM hinders diffusion in the biofilm. The aim of the study was to determine via RT-qPCR the dynamics of expression of S. mutans genes associated with LTA (dltABCD, SMU_775c), eDNA (lytST, lrgAB, ccpA) and exopolysaccharides (gtfBCD, gbpB, dexA) metabolism, during ECM development in mixed-species biofilm by time-lapse studies. Mixed-species biofilms of S. mutans UA159 (parental strain) or ΔgtfB (mutant with deletion of the gene gtfB), Actinomyces naeslundii ATCC 12104 and Streptocsoccus gordonii DL-1 were formed onto saliva-coated hydroxyapatite discs. These biofilms were cultivated at 37°C and 5% CO2 in triptone with yeast extract containing saliva 25% and alternating 0.1% sucrose (scarcity) and 0.5% sucrose + 1% starch (abundance). The pH of the spent media remained acid during the experimental periods, and was lower after prolonged incubation periods. All genes were expressed at distinct levels in both biofilm types at all ages (29, 43, 55, 67, 79, 91, 103 and 115 hours). Genes associated with LTA and EPS were expressed in a similar way, having higher expression levels two hours after providing carbohydrates. Both strains presented similar expression profile, with higher expression levels in UA159 biofilm. Genes associated with eDNA presented a different expression dynamic between the parental strain and ΔgtfB strain. In UA159 biofilms lrgAB genes were highly expressed at 29h, and did not appear to be related with lytST genes, while in ΔgtfB biofilms an inverse relationship between lytS and lrgAB was detect at times when the medium was “fresh” (two hours after medim change). For both biofilms, the lytT gene presented the lowest expression levels, without having differences between the periods. The product of ccpA gene activates or represses the expression of certain genes as a response to the carbohydrates availability, this phenomenon more evident in UA159 biofilm. Therefore, the deletion of gtfB influences the expression dynamics of the evaluated genes, decreasing both the magnitude of the expression of genes associated with EPS, and changing the expression profile of associated genes with the presence of eDNA in the matrix.
Dental caries represents the most prevalent human disease worldwide, its etiology is biofilm-diet dependent. Biofilms are highly dynamic and structured communities of microbial cells that are enmeshed in a three-dimensional extracellular matrix (ECM). This structure acts as a diffusion-limiting barrier providing stability and protection to the microorganisms. Streptococcus mutans is acidogenic, aciduric, and orchestrates the biofilm build-up process. It modulates the biofilm’s virulence by producing lipoteichoic acids (LTA), extracellular DNA (eDNA) and exopolysaccharides (EPS), thereby promoting microbial adhesion and cohesion. The resulting ECM hinders diffusion in the biofilm. The aim of the study was to determine via RT-qPCR the dynamics of expression of S. mutans genes associated with LTA (dltABCD, SMU_775c), eDNA (lytST, lrgAB, ccpA) and exopolysaccharides (gtfBCD, gbpB, dexA) metabolism, during ECM development in mixed-species biofilm by time-lapse studies. Mixed-species biofilms of S. mutans UA159 (parental strain) or ΔgtfB (mutant with deletion of the gene gtfB), Actinomyces naeslundii ATCC 12104 and Streptocsoccus gordonii DL-1 were formed onto saliva-coated hydroxyapatite discs. These biofilms were cultivated at 37°C and 5% CO2 in triptone with yeast extract containing saliva 25% and alternating 0.1% sucrose (scarcity) and 0.5% sucrose + 1% starch (abundance). The pH of the spent media remained acid during the experimental periods, and was lower after prolonged incubation periods. All genes were expressed at distinct levels in both biofilm types at all ages (29, 43, 55, 67, 79, 91, 103 and 115 hours). Genes associated with LTA and EPS were expressed in a similar way, having higher expression levels two hours after providing carbohydrates. Both strains presented similar expression profile, with higher expression levels in UA159 biofilm. Genes associated with eDNA presented a different expression dynamic between the parental strain and ΔgtfB strain. In UA159 biofilms lrgAB genes were highly expressed at 29h, and did not appear to be related with lytST genes, while in ΔgtfB biofilms an inverse relationship between lytS and lrgAB was detect at times when the medium was “fresh” (two hours after medim change). For both biofilms, the lytT gene presented the lowest expression levels, without having differences between the periods. The product of ccpA gene activates or represses the expression of certain genes as a response to the carbohydrates availability, this phenomenon more evident in UA159 biofilm. Therefore, the deletion of gtfB influences the expression dynamics of the evaluated genes, decreasing both the magnitude of the expression of genes associated with EPS, and changing the expression profile of associated genes with the presence of eDNA in the matrix.
Descrição
Palavras-chave
Biofilmes, Cárie dentária, Expressão gênica, Matriz extracelular, Streptococcus mutans, Biofilms, Dental caries, Extracellular matrix, Gene expression