Influência de alterações genéticas, do fluconazol e de enzimas hidrolíticas na matriz extracelular de biofilmes de candida susceptível e resistente a fluconazol
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Data
2017-08-04
Autores
Panariello, Beatriz Helena Dias [UNESP]
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Universidade Estadual Paulista (Unesp)
Resumo
Biofilmes formados por Candida estão relacionados a infecções bucais, como a candidíase. Embora a resistência do biofilme seja multifatorial, a proteção exercida por sua matriz extracelular (MEC) é importante para os altos níveis de resistência a drogas antifúngicas. O conhecimento dos princípios estruturais da MEC possibilita maior compreensão de como atuar para desorganizá-la e melhorar a difusão de agentes antifúngicos para que atinjam mais eficientemente o biofilme, além de, futuramente, possibilitar o desenvolvimento de terapias mais eficazes para o controle da formação de biofilmes. Sendo assim, os objetivos principais deste estudo foram: (1) verificar a influência da inativação de genes envolvidos na filamentação (EFG1 e TEC1) em características estruturais dos biofilmes e na produção de componentes da MEC; (2) verificar a influência do fluconazol (FLZ) na MEC de biofilmes de Candida albicans ATCC 90028 (susceptível a fluconazol- CaS), C. albicans ATCC 96901 (resistente a fluconazol- CaR), Candida glabrata ATCC 2001 (susceptível a fluconazol- CgS) e C. glabrata ATCC 200918 (resistente a fluconazol- CgR) e (3) estudar a ação de enzimas hidrolíticas (DNase, Dextranase e β-glucanase individualmente ou em diferentes combinações) sobre a MEC de biofilmes de CaS e CaR. Biofilmes maduros (48 horas) foram analisados através de contagem de unidades formadoras de colônia (ufc/mL), peso seco total, peso seco insolúvel e proteínas insolúveis. Os componentes da MEC- polissacarídeos solúveis em álcali (ASPs), polissacarídeos solúveis em água (WSPs), DNA extracelular (eDNA) e proteínas solúveis- foram quantificados. No estudo 1, foi observado que o conteúdo de ASPs é significativamente maior em cepa parental de C. albicans em comparação com as cepas mutantes Δ/Δ efg1 e Δ/Δ tec1, o que indica que a produção de ASPs pode estar relacionada à morfologia celular filamentosa em C. albicans. No estudo 2, observou-se que as biomassas totais e WSPs foram significativamente reduzidos pelo FLZ na MEC de CaS, CaR, CgS e CgR, mas as quantidades de eDNA e proteínas não foram influenciadas pela presença de FLZ nem pelo tipo de cepa. O FLZ interferiu na morfologia celular e na estrutura dos biofilmes, reduzindo a formação de hifas nos biofilmes de CaS e CaR e diminuindo o número de células nos biofilmes de CgS e CgR. No estudo 3, observou-se que exposição de biofilmes maduros à DNase por 5 minutos reduziu o conteudo de eDNA, polissacarídeos e proteínas solúveis da MEC de CaS e CaR, sendo um promissor adjuvante para terapias antibiofilme. A redução de polissacarídeos extracelulares e do conteúdo de proteínas pela DNase indicam que esses componentes estão interligados ao eDNA na MEC de CaS e CaR. Portanto, células filamentosas têm tendência de produzirem mais exopolissacarídeos, e estes componentes estão interligados ao eDNA e a proteínas solúveis na MEC de biofilmes de C. albicans. Para reduzir componentes da matriz e desorganizar a estrutura formada por eDNA-exopolissacarídeos-proteínas, a aplicação de enzima DNase por 5 min em biofilmes maduros de C. albicans se mostrou eficaz
Biofilms formed by Candida are related to bucal infections, such as candidiasis. Although the biofilm resistance is multifactorial, the protection exerted by its extracellular matrix (ECM) is essential for its high levels of resistance to antifungals. The knowledge of the structural principles of the ECM permits a better understanding of how to disorganize the ECM and improve the diffusion of antifungal drugs to reach the biofilm. Moreover, the study of the ECM may enable the development of more effective therapies to control biofilm formation. Thus, the main objectives of this study were: (1) to verify the influence of the inactivation of genes involved in filamentation and structural characteristics of the biofilms (EFG1 and TEC1) on the production of ECM components; (2) to verify the influence of fluconazole (FLZ) on the biofilms’ ECM of Candida albicans ATCC 90028 (fluconazole-susceptible: CaS), C. albicans ATCC 96901 (fluconazole-resistant: CaR), Candida glabrata ATCC 2001 (fluconazolesusceptible: CgS) and C. glabrata ATCC 200918 (fluconazole-resistant: CgR) and (3) to study the action of hydrolytic enzymes (DNase, Dextranase and β-glucanase individually or in different combinations) on the ECM of CaS and CaR biofilms. Mature biofilms (48 hours) were analyzed by colony counting forming units (cfu/mL), total dry weight, insoluble dry-weight and total proteins. ECM components- alkali-soluble polysaccharides (ASPs), water-soluble polysaccharides (WSPs), extracellular DNA (eDNA) and soluble proteins- were quantified. In study 1, it was observed that ASPs content is significantly higher in C. albicans parental strain compared to the mutant strains Δ/Δ efg1 and Δ/Δ tec1, indicating that the production of ASPs may be related to the filamentous cell morphology in C. albicans. In study 2, it was observed that the total biomasses and WSPs were significantly reduced by FLZ in the ECM of CaS, CaR, CgS and CgR, but the amounts of eDNA and proteins were not influenced by the presence of FLZ nor by type of strain. FLZ interfered on the cellular morphology and structure of biofilms, reducing hyphae formation in CaS and CaR biofilms and reducing the number of cells in CgS and CgR biofilms. The study 3 demonstrated that exposure of mature biofilms to DNase for 5 minutes reduced the eDNA, polysaccharides and soluble proteins of the ECM of CaS and CaR, being a promising adjuvant for antibiofilm therapies. The reduction of extracellular polysaccharides and soluble proteins by DNase indicates that these components are intertwined to eDNA in the ECM of CaS and CaR. Therefore, filamentous cells tend to produce more exopolysaccharides, and these components are intertwined to eDNA and soluble proteins in the ECM of C. albicans biofilms. To reduce matrix components and disrupt the structure formed by eDNA-exopolysaccharide-proteins, 5 min exposure of mature biofilms to DNase showed to be effective
Biofilms formed by Candida are related to bucal infections, such as candidiasis. Although the biofilm resistance is multifactorial, the protection exerted by its extracellular matrix (ECM) is essential for its high levels of resistance to antifungals. The knowledge of the structural principles of the ECM permits a better understanding of how to disorganize the ECM and improve the diffusion of antifungal drugs to reach the biofilm. Moreover, the study of the ECM may enable the development of more effective therapies to control biofilm formation. Thus, the main objectives of this study were: (1) to verify the influence of the inactivation of genes involved in filamentation and structural characteristics of the biofilms (EFG1 and TEC1) on the production of ECM components; (2) to verify the influence of fluconazole (FLZ) on the biofilms’ ECM of Candida albicans ATCC 90028 (fluconazole-susceptible: CaS), C. albicans ATCC 96901 (fluconazole-resistant: CaR), Candida glabrata ATCC 2001 (fluconazolesusceptible: CgS) and C. glabrata ATCC 200918 (fluconazole-resistant: CgR) and (3) to study the action of hydrolytic enzymes (DNase, Dextranase and β-glucanase individually or in different combinations) on the ECM of CaS and CaR biofilms. Mature biofilms (48 hours) were analyzed by colony counting forming units (cfu/mL), total dry weight, insoluble dry-weight and total proteins. ECM components- alkali-soluble polysaccharides (ASPs), water-soluble polysaccharides (WSPs), extracellular DNA (eDNA) and soluble proteins- were quantified. In study 1, it was observed that ASPs content is significantly higher in C. albicans parental strain compared to the mutant strains Δ/Δ efg1 and Δ/Δ tec1, indicating that the production of ASPs may be related to the filamentous cell morphology in C. albicans. In study 2, it was observed that the total biomasses and WSPs were significantly reduced by FLZ in the ECM of CaS, CaR, CgS and CgR, but the amounts of eDNA and proteins were not influenced by the presence of FLZ nor by type of strain. FLZ interfered on the cellular morphology and structure of biofilms, reducing hyphae formation in CaS and CaR biofilms and reducing the number of cells in CgS and CgR biofilms. The study 3 demonstrated that exposure of mature biofilms to DNase for 5 minutes reduced the eDNA, polysaccharides and soluble proteins of the ECM of CaS and CaR, being a promising adjuvant for antibiofilm therapies. The reduction of extracellular polysaccharides and soluble proteins by DNase indicates that these components are intertwined to eDNA in the ECM of CaS and CaR. Therefore, filamentous cells tend to produce more exopolysaccharides, and these components are intertwined to eDNA and soluble proteins in the ECM of C. albicans biofilms. To reduce matrix components and disrupt the structure formed by eDNA-exopolysaccharide-proteins, 5 min exposure of mature biofilms to DNase showed to be effective
Descrição
Palavras-chave
Candida, Biofilmes, Matriz extracelular, Resistência a medicamentos, Fluconazol, Mutação, Candida, Biofilms, Extracellular matrix, Drug resistance, Fluconazole, Mutation