Parte III – Modificações na forma de agregados devido a introdução de molécula sinalizadora
6. Conclusões
Nossos resultados mostram que o EPS está presente como cobertura celular em estágios muito precoces de adesão celular de X.fastidiosa subsp. pauca. Procedimentos de lavagem não foram capazes de eliminar completamente EPS, indicando uma forte interação com a membrana celular bacteriana. Medições de espectroscopia de força em células individuais e pequenos biofilmes mostram que o stiffness do sistema EPS / célula diminui com o tempo de crescimento e que as regiões polares são menos rígidas do que o corpo bacteriano. Estes resultados concordam bem com a hipótese de mudanças locais na composição de EPS de X.fastidiosa em regiões de tamanho micrométrico, sugeridas a partir de estudos anteriores com medidas de potencial de superfície [13].
A flexibilidade associada com materiais mais macios na região do polo da célula pode explicar a estabilidade do movimento oscilante de células isoladas, aderidas verticalmente, observadas nos experimentos em ambientes microfluídicos. Ao menor valor de stiffness da matriz EPS para biofilmes com maior tempo de crescimento também explica o caráter maleável da matriz, que permite o transporte celular e o eventual desprendimento de EPS e células, observado nos microcanais. Estas últimas experiências foram realizadas sob fluxos simulando condições de xilema, e também corroboram a interpretação de resultados anteriores de crescimento bacteriano estático [13].
Além disso, este tipo de investigação poderia ser utilizado para identificar marcadores que desencadeiam a variação da composição de EPS e assim diferentes estágios para a formação de biofilmes. A resposta elástica observada neste trabalho indica uma maior flexibilidade nos pontos de ligação da superfície celular, o que pode ser importante para a adaptação célula- hospedeiro, afetando desde a transmissão celular de/para o vetor de insetos até a migração celular através de vasos de xilema em plantas.
Quanto aos experimentos em ambiente microfluídicos verificamos que houve um aumento na densidade bacteriana devido ao confinamento celular promovido pelo microambiente. Além disso, utilizando microcanais tratados quimicamente com a adesina XadA1, presente nos estágios iniciais de adesão bacteriana, identificamos seu papel como moduladora da adesão bacteriana à superfície e na interação célula/célula. Quantificamos a força de adesão associada à sua presença na superfície; foram observados valores 2 vezes maiores se comparados à superfície controle. Verificamos ainda em estudos preliminares que a presença de DSF afeta a
forma dos agregados bacterianos e promove um aumento da área coberta por células individuais, agregados e biofilmes, podendo favorecer de alguma maneira a adesão bacteriana.
Diante dos resultados apresentados nesta tese, poderiam ser feitas algumas modificações nas condições experimentais para extrair mais informações sobre o sistema analisado. Poderíamos por exemplo destacar, o uso de microscópio confocal com z-stack para monitorar a forma dos agregados durante os experimentos nos microcanais impressos. Porém, além de uma melhor caracterização do perfil difusivo nos microcanais acoplados por membrana, são também necessárias alterações na forma como é feita a adesão vidro/PLA que permitam maior durabilidade dos dispositivos e consequentemente uma maior janela de monitoramento experimental.
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