Avaliação, desenho e padronização de primers para genes de virulência de Candida albicans e sua expressão em isolados clínicos submetidos à terapias antifúngicas

Autor(es)

Gabriela Caroline Alonso

• Tipo de documento: Dissertação de Mestrado
• Imprenta: Araraquara. 2018-03-07.
• Instituição: Universidade Estadual Paulista (Unesp). Faculdade de Odontologia. Araraquara
• Data de defesa: 2017-07-07
• Orientador: Ana Cláudia Pavarina; Marlise Inês Klein

Resumo

This study evaluated longitudinally the expression of Candida albicans virulence genes on clinical isolates from patients with oral candidiasis treated with Antimicrobial Photodynamic Therapy (aPDT) or Nystatin (NIS). First, specificity of primers from the literature and newly designed primers for C. albicans virulence genes ALS1, CAP1, CAT1, EFG1, HWP1, LIP3, PLB1, SAP1, SAP4, SOD1, SOD5 and ACT1 (control gene) was evaluated through in silico and in vitro analyzes. For in silico analysis, a Pubmed search was performed for studies with primer sequences that evaluated gene expression of C. albicans. Then, the homology of these primers was checked (BLAST and ClustalW2) as well as the presence of secondary structures (Mfold). New primers were designed (Beacon Designer ™) from sequences obtained from the "Candida Genome Database". The primers were synthesized and tested in vitro by PCR using a panel of genomic DNA from different Candida species, with their products visualized on agarose gel. qPCR reactions were performed to determine primers’ optimal concentration and efficiency. For gene expression analysis, patients were submitted to aPDT (6 sessions with applications of Photoditazine™ photosensitizer (200 mg/mL) on the palate and dentures for 20 minutes with subsequent application of LED (660 nm - 50 J / cm² )] or conventional treatment [1 minute mouthwash with 1 mL of Nystatin solution (100.000 UI/ mL) four times a day for 15 days]. Microbiological cultures were taken at the initial (before treatment), final (post-treatment) and 60 days after treatment initiation. The samples were plated in CHROMagar medium and the colonies (C. albicans) were submitted to RNA extraction and purification. The cDNA was synthesized and RT-qPCR technique was performed. Expression data were analyzed by Analysis of Variance for Mixed Repeated Measures (α = 0.05). The literature primers for the SAP1 and HWP1 genes were specific for C. albicans while the SOD1 gene reacted with C. albicans and Candida dubliniensis. The designed primers for ACT1, ALS1 and HWP1 genes were detected only in C. albicans, while the primers for CAP1, CAT1, EFG1, LIP3 and PLB1 genes were detected in C. albicans and C. dubliniensis. All primers presented ideal Melt Curves, correlation coefficient of ≅1 and efficiency between 90-110%, with slope ≅-3.3. The data showed that the gene expression of CAT1 gene was higher in the final culture compared to the initial culture for both groups (p = 0,041). Expression of the LIP3 gene was significantly reduced from initial to final culture, regardless of treatment (p = 0,039). The SOD1 gene had its expression decreased between the cultures, regardless of the treatment (p = 0,021). The PLB1 and ACT1 genes expression was higher in the aPDT group, independent of the cultures (p < 0,01 and p = 0,046, respectively). For the ALS1, CAP1, EFG1, HWP1 and SAP1 genes, there was no statistical difference between the cultures, independent of the treatment, which did not influenced the expression of such genes (p > 0,05). Therefore, the data show that the primers are standardized for gene expression analyzes from clinical samples and there are no significant differences on virulence gene expression of clinical isolates of C. albicans from patients treated with aPDT when compared to those treated with NIS, except for PLB1 and ACT1 genes. (AU)

• Processo FAPESP: 15/13409-0 - Expressão de genes de virulência de isolados clínicos de Candida albicans submetidos à Terapia Fotodinâmica Antimicrobiana
• Beneficiário: Gabriela Caroline Alonso
• Linha de fomento: Bolsas no Brasil - Mestrado