Expressão de genes relacionados a virulência e envolvidos na síntese de ergosterol de Candida albicans resistente a fluconazol submetidos à terapia fotodinâmica associada ao antifúngico
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Data
2019-05-22
Autores
Jordão, Cláudia Carolina
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Universidade Estadual Paulista (Unesp)
Resumo
O estudo tem como objetivo avaliar a susceptibilidade ao fluconazol e a expressão de genes de virulência e envolvidos na síntese de ergosterol de cepa de Candida albicans resistente a fluconazol (ATCC 96901) presentes nas línguas de camundongos submetidos à aPDT associada a Nistatina. Para isso, colônias recuperadas de animais submetidos ao tratamento com aPDT e Nistatina (NIS) bem como a associação (NIS+aPDT e aPDT+NIS), foram armazenadas em glicerol 50% e utilizadas para o Teste de Concentração Inibitória Mínima (CIM) e Concentração Fungicida Mínima (CFM). Além disso, as línguas dos animais submetidos aos tratamentos foram armazenadas em RNAlater (RNAlater Tissue Collection: RNA Stabilization Solution Protocol Ambion®) em freezer -80°C para avaliação da expressão gênica. Para realização do teste CIM, as cepas foram reativadas em placas de Ágar Sabouraud Dextrose (SDA), cultivadas em estufa 35°C por 48 h e avaliadas seguindo o método de microdiluição em caldo (CLSI) com algumas modificações. Foram realizadas diluições seriadas de fluconazol (variando de 2 a 1024 μg/ml) em meio RPMI 1640 (2 x concentrado), em suspensões (0,5x103 a 2,5x103 UFC/mL). As placas foram incubadas a 35°C e observadas quanto à presença ou ausência de crescimento após 24 h, além disso, foi realizado plaqueamento (CFM). Foram consideradas as menores concentrações de fluconazol que resultaram em inibição de pelo menos 50% do crescimento. A quantificação da expressão gênica foi realizada por meio da técnica quantitativa de Transcrição Reversa da reação em cadeia de polimerase (RT-qPCR) utilizando primers específicos para os genes de virulência (ALS1; HWP1; EFG1; CAP1; CAT1; SOD1; SAP1; PLB1 e LIP3) e genes envolvidos na síntese do ergosterol (ERG1; ERG11; ERG3 e ERG25). Novos primers para os genes ERGs foram desenhados (Oligo Software™) a partir de seqüências obtidas do "Pubmed". Os primers foram sintetizados e testados in vitro pela técnica de PCR utilizando um painel de DNA genômico de diferentes espécies de Candida, com seus produtos visualizados em gel de agarose. Reações de qPCR foram realizadas para determinar a concentração ótima e a eficiência dos primers. Para análise da expressão gênica, as línguas foram submetidas à extração e purificação de RNA. O cDNA foi sintetizado e a técnica de RT-qPCR realizada. Os dados de CFM e expressão foram analisados por Análise de Variância (α = 0,05). Os resultados do CFM pós CIM foram avaliados por ANOVA seguida do pós-teste de Tukey (α=0,05). Foi verificado que, independente do tratamento aplicado e do momento da coleta (24h ou 7 dias após tratamento), as cepas recuperadas foram semelhantes ao grupo P-L-(animais sem tratamento). Demonstrando que não houve modificação do perfil de resistência da cepa ao fluconazol nas concentrações testadas. Em relação a expressão gênica, os primers delineados não foram específicos somente para C. albicans. No presente estudo, foi observado que os genes ERG1, ERG3, ERG11 e ERG25 apresentaram comportamento semelhante, com redução da expressão gênica nos grupos onde foram realizados os tratamentos em comparação com o grupo controle (P-L-). Os resultados demonstraram que os tratamentos reduziram a expressão dos genes ALS1, EFG1, CAP1, LIP3, SAP1 e SOD1 em todos os grupos experimentais avaliados em relação ao controle (P-L-). Quando utilizada a terapia combinada (aPDT+NIS, NIS+aPDT), houve redução mais acentuada na expressão dos genes ALS1, LIP3, SAP1 e SOD1. A expressão do gene EFG1, foi estatisticamente semelhante em todos os grupos de tratamento e menor que no grupo controle (P-L-). Para os genes CAT1 e SOD1, não foi observada diferença estatística entre os grupos aPDT e aPDT+NIS. Para os genes CAP1, EFG1, HWP1 e LIP3, os grupos P-L+ e aPDT não apresentaram diferença estatística entre si. Para os genes CAP1, EFG1 e HWP1 não houve diferença estatística entre os grupos de tratamento NIS e os que foram avaliados a associação das terapias (NIS+aPDT e aPDT+NIS). Não houve expressão detectada para o gene PLB1. A associação de terapias não alterou a susceptibilidade fúngica ao fluconazol e reduziu a expressão dos genes ERG1, ERG3, ERG11, ERG25, ALS1, EFG1, SAP1, LIP3, CAP1 e SOD1.
The aim of this study was to evaluate the susceptibility to fluconazole and the expression of virulence genes and involved in ergosterol synthesis of fluconazole resistant strain of Candida albicans (ATCC 96901) present in the mouse’s thongue submitted to aPDT associated with Nystatin. For this, colonies recovered from animals submitted to treatment with aPDT and Nystatin (NIS) as well as their association (NIS + aPDT and aPDT + NIS) were stored in 50% glycerol and used for the Minimum Inhibitory Concentration Test (MIC) and Minimum Fungicidal Concentration (MFC). In addition, part of the tongue of the treated animals were stored in RNAlater (RNAlater Tissue Collection: RNA Stabilization Solution Protocol Ambion®) in -80 °C freezer for evaluation of gene expression. To accomplish the MIC test, the strains were reactivate in Sabouraud Dextrose Agar (SDA) plates, grown in a 35 °C oven for 48 h and evaluated using the broth microdilution method (CLSI) with some modifications. Serial dilutions of fluconazole (ranging from 2 to 1024 μg / ml) in RPMI 1640 medium (2x concentrate) were performed in suspensions (0.5x103 to 2.5x103 CFU / ml). The plates were incubated at 35 ° C and observed for presence or absence of growth after 24 h, plating was also performed (CFM). The lowest concentrations of fluconazole that resulted in inhibition of at least 50% growth were consider. The quantification of the gene expression was performed using quantitative Reverse Transcription-quantitative polymerase chain reaction (RT-qPCR) using specific primers for the virulence genes (ALS1; HWP1; EFG1; CAP1; CAT1; SOD1; SAP1; PLB1 and LIP3) and genes involved in the synthesis of ergosterol (ERG1; ERG11; ERG3 and ERG25). New primers for the ERG genes were designed (Oligo Software ™) from sequences obtained from "Pubmed". The primers were design and tested in vitro by the PCR technique using a panel of genomic DNA from different Candida species, with their products visualized on agarose gel. Reactions of qPCR were performe to determine the optimum concentration and efficiency of the primers. For the analysis of the gene expression, the tongue were submitt to the extraction and purification of RNA. The cDNA was synthesized and the RT-qPCR technique was performed. The CFM and expression data were analyzed by Analysis of Variance (α = 0.05). The results of the CFM before MIC were evaluated by ANOVA followed by the Tukey post-test (α = 0.05). Regardless of the treatment applied and the time of collection (24h or 7 days after treatment), the recovered strains showed a similar behavior to the P-L- group. Demonstrating that there was no change in the resistance profile of the strain to fluconazole at the concentrations tested. Regarding the q-PCR data, the designed primers were not specific only for C. albicans. All primers presented ideal Melt Curves, correlation coefficient ≅1 and efficiency between 90-110%, with slope ≅-3.3. In the present study, the expression of ERG1, ERG3, ERG11 and ERG25 genes presented similar behavior, with reduction expression in the groups that the treatments were performe in comparison with the control group (P-L-). The results showed that the treatments reduced the expression of ALS1, EFG1, CAP1, LIP3, SAP1 and SOD1 genes in all the experimental groups evaluated in relation to the control (P-L-). When combined therapy (aPDT + NIS, NIS + aPDT) was use, there was pronounce reduction in the expression of ALS1, LIP3, SAP1 and SOD1 genes. EFG1 gene expression was statistically similar in all treatment groups and lower than in the control group (P-L-). For the CAT1 and SOD1 genes, no statistical difference was observe between aPDT and aPDT + NIS groups. For the CAP1, EFG1, HWP1 and LIP3 genes, the P-L + and aPDT groups showed no statistical difference between them. For the CAP1, EFG1 and HWP1 genes, there was no statistical difference between the NIS treatment groups and those that evaluated the association of the therapies (NIS + aPDT and aPDT + NIS). There was no expression detected for the PLB1 gene. The association of therapy did not change the fungal susceptibility to fluconazole and reduced expression of genes ERG1, ERG3, ERG11, ERG25, ALS1, EFG1, SAP1, LIP3, CAP1 and SOD1.
The aim of this study was to evaluate the susceptibility to fluconazole and the expression of virulence genes and involved in ergosterol synthesis of fluconazole resistant strain of Candida albicans (ATCC 96901) present in the mouse’s thongue submitted to aPDT associated with Nystatin. For this, colonies recovered from animals submitted to treatment with aPDT and Nystatin (NIS) as well as their association (NIS + aPDT and aPDT + NIS) were stored in 50% glycerol and used for the Minimum Inhibitory Concentration Test (MIC) and Minimum Fungicidal Concentration (MFC). In addition, part of the tongue of the treated animals were stored in RNAlater (RNAlater Tissue Collection: RNA Stabilization Solution Protocol Ambion®) in -80 °C freezer for evaluation of gene expression. To accomplish the MIC test, the strains were reactivate in Sabouraud Dextrose Agar (SDA) plates, grown in a 35 °C oven for 48 h and evaluated using the broth microdilution method (CLSI) with some modifications. Serial dilutions of fluconazole (ranging from 2 to 1024 μg / ml) in RPMI 1640 medium (2x concentrate) were performed in suspensions (0.5x103 to 2.5x103 CFU / ml). The plates were incubated at 35 ° C and observed for presence or absence of growth after 24 h, plating was also performed (CFM). The lowest concentrations of fluconazole that resulted in inhibition of at least 50% growth were consider. The quantification of the gene expression was performed using quantitative Reverse Transcription-quantitative polymerase chain reaction (RT-qPCR) using specific primers for the virulence genes (ALS1; HWP1; EFG1; CAP1; CAT1; SOD1; SAP1; PLB1 and LIP3) and genes involved in the synthesis of ergosterol (ERG1; ERG11; ERG3 and ERG25). New primers for the ERG genes were designed (Oligo Software ™) from sequences obtained from "Pubmed". The primers were design and tested in vitro by the PCR technique using a panel of genomic DNA from different Candida species, with their products visualized on agarose gel. Reactions of qPCR were performe to determine the optimum concentration and efficiency of the primers. For the analysis of the gene expression, the tongue were submitt to the extraction and purification of RNA. The cDNA was synthesized and the RT-qPCR technique was performed. The CFM and expression data were analyzed by Analysis of Variance (α = 0.05). The results of the CFM before MIC were evaluated by ANOVA followed by the Tukey post-test (α = 0.05). Regardless of the treatment applied and the time of collection (24h or 7 days after treatment), the recovered strains showed a similar behavior to the P-L- group. Demonstrating that there was no change in the resistance profile of the strain to fluconazole at the concentrations tested. Regarding the q-PCR data, the designed primers were not specific only for C. albicans. All primers presented ideal Melt Curves, correlation coefficient ≅1 and efficiency between 90-110%, with slope ≅-3.3. In the present study, the expression of ERG1, ERG3, ERG11 and ERG25 genes presented similar behavior, with reduction expression in the groups that the treatments were performe in comparison with the control group (P-L-). The results showed that the treatments reduced the expression of ALS1, EFG1, CAP1, LIP3, SAP1 and SOD1 genes in all the experimental groups evaluated in relation to the control (P-L-). When combined therapy (aPDT + NIS, NIS + aPDT) was use, there was pronounce reduction in the expression of ALS1, LIP3, SAP1 and SOD1 genes. EFG1 gene expression was statistically similar in all treatment groups and lower than in the control group (P-L-). For the CAT1 and SOD1 genes, no statistical difference was observe between aPDT and aPDT + NIS groups. For the CAP1, EFG1, HWP1 and LIP3 genes, the P-L + and aPDT groups showed no statistical difference between them. For the CAP1, EFG1 and HWP1 genes, there was no statistical difference between the NIS treatment groups and those that evaluated the association of the therapies (NIS + aPDT and aPDT + NIS). There was no expression detected for the PLB1 gene. The association of therapy did not change the fungal susceptibility to fluconazole and reduced expression of genes ERG1, ERG3, ERG11, ERG25, ALS1, EFG1, SAP1, LIP3, CAP1 and SOD1.
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Palavras-chave
Fotoquimioterapia, Candida albicans, Expressão Gênica, Photochemotherapy, Candida albicans, Gene Expression