Influência de alterações genéticas, do fluconazol e de enzimas hidrolíticas na matriz extracelular de biofilmes de candida susceptível e resistente a fluconazol

Autor(es)

Beatriz Helena Dias Panariello

• Tipo de documento: Tese de Doutorado
• Imprenta: Araraquara. 2018-01-26.
• Instituição: Universidade Estadual Paulista (Unesp). Faculdade de Odontologia. Araraquara
• Data de defesa: 2017-08-04
• Orientador: Ana Cláudia Pavarina; Marlise Inêz Klein

Resumo

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 (AU)

• Processo FAPESP: 14/18804-1 - Matriz extracelular de biofilmes de Candida albicans e Candida glabrata susceptíveis e resistentes ao fluconazol: Caracterização, ação de enzimas hidrolíticas e eficácia da associação das enzimas à Terapia Fotodinâmica.
• Beneficiário: Beatriz Helena Dias Panariello
• Modalidade de apoio: Bolsas no Brasil - Doutorado