Por muitos anos o fluconazol tem sido uma opção usual para tratamento de infecções por Candida. Entretanto, o uso indiscriminado desta terapia antimicótica tem favorecido o surgimento de microrganismos resistentes. A redução da afinidade da enzima alvo dos antifúngicos, 14--demetilase (ERG11p), tem sido descrita como um importante mecanismo de resistência, caracterizado por mutações em seu gene codificante ERG11. Neste estudo, foi investigada a suscetibilidade ao fluconazol de 87 isolados de C. albicans, C. tropicalis, C. parapsilosis, C. krusei e C. glabrata, com valores de MIC determinados através do método de microdiluição em caldo M27-A3 (CLSI, 2008); verificou-se que dezessete isolados apresentavam decréscimo da suscetibilidade ao fluconazol. A triagem de mutações foi realizada através da amplificação de quatro regiões do gene ERG11 com primers específicos delineados neste estudo, para cada espécie de Candida, seguida de análise pela técnica de eletroforese SSCP e seqüenciamento automatizado. Foram identificadas 217 mutações, incluindo 185 silenciosas e 32 por troca de sentido (que altera o aminoácido resultante). Estas últimas foram observadas em 19 isolados e 17 resíduos distintos, sendo 7 deles ainda não descritos anteriormente: L321F em C. albicans; K53M em C. krusei; Y221F, K344T, V362M e R371S em C. tropicalis; e R398I em C. parapsilosis. Confrontando os resultados entre a técnica de eletroforese SSCP e seqüenciamento automatizado, não houve associação direta entre as mudanças na migração eletroforética e alterações na seqüência nucleotídica do gene ERG11. Sugerimos que a freqüência elevada de alterações no gene ERG11 de Candida deve ser considerada no design de novos fármacos que visem a enzima ERG11p / For many years, fluconazole has been a usual option for treatment of Candida infections. However, the indiscriminate use of antimycotic therapy has favored the emergence of resistant organisms. The decrease in the affinity of the enzyme target of antifungal agents, 14--demethylase (ERG11p), has been described as an important mechanism of resistance, characterized by mutations in its encoding gene ERG11. In this study, we investigated the susceptibility to fluconazole of 87 strains of C. albicans, C. tropicalis, C. parapsilosis, C. krusei and C. glabrata with MIC values determined by broth microdilution M27-A3 (CLSI, 2008), found that seventeen isolates showed decreased susceptibility to fluconazole. Mutation screening was performed by the amplification of four regions of the ERG11 gene with specific primers designed in this study for each Candida species, followed by electrophoresis SSCP analysis and sequencing. We identified 217 mutations, including 185 silent mutations and 32 missense mutations. Missense mutations were observed in 19 isolates and 17 distinct residues, 7 of them still not described earlier: L321F in C. albicans, K53M in C. krusei; Y221F, K344T, V362M and R371S in C. tropicalis, and R398I in C. parapsilosis. Comparing the results between SSCP electrophoresis technique and sequencing, there was no direct association between changes in electrophoretic migration and changes in the nucleotide sequence of the ERG11 gene. We suggest that the high frequency of changes in ERG11 gene of Candida should be considered in the design of new drugs targeting the enzyme ERG11p

Por muitos anos o fluconazol tem sido uma opção usual para tratamento de infecções por Candida. Entretanto, o uso indiscriminado desta terapia antimicótica tem favorecido o surgimento de microrganismos resistentes. A redução da afinidade da enzima alvo dos antifúngicos, 14--demetilase (ERG11p), tem sido descrita como um importante mecanismo de resistência, caracterizado por mutações em seu gene codificante ERG11. Neste estudo, foi investigada a suscetibilidade ao fluconazol de 87 isolados de C. albicans, C. tropicalis, C. parapsilosis, C. krusei e C. glabrata, com valores de MIC determinados através do método de microdiluição em caldo M27-A3 (CLSI, 2008); verificou-se que dezessete isolados apresentavam decréscimo da suscetibilidade ao fluconazol. A triagem de mutações foi realizada através da amplificação de quatro regiões do gene ERG11 com primers específicos delineados neste estudo, para cada espécie de Candida, seguida de análise pela técnica de eletroforese SSCP e seqüenciamento automatizado. Foram identificadas 217 mutações, incluindo 185 silenciosas e 32 por troca de sentido (que altera o aminoácido resultante). Estas últimas foram observadas em 19 isolados e 17 resíduos distintos, sendo 7 deles ainda não descritos anteriormente: L321F em C. albicans; K53M em C. krusei; Y221F, K344T, V362M e R371S em C. tropicalis; e R398I em C. parapsilosis. Confrontando os resultados entre a técnica de eletroforese SSCP e seqüenciamento automatizado, não houve associação direta entre as mudanças na migração eletroforética e alterações na seqüência nucleotídica do gene ERG11. Sugerimos que a freqüência elevada de alterações no gene ERG11 de Candida deve ser considerada no design de novos fármacos que visem a enzima ERG11p / For many years, fluconazole has been a usual option for treatment of Candida infections. However, the indiscriminate use of antimycotic therapy has favored the emergence of resistant organisms. The decrease in the affinity of the enzyme target of antifungal agents, 14--demethylase (ERG11p), has been described as an important mechanism of resistance, characterized by mutations in its encoding gene ERG11. In this study, we investigated the susceptibility to fluconazole of 87 strains of C. albicans, C. tropicalis, C. parapsilosis, C. krusei and C. glabrata with MIC values determined by broth microdilution M27-A3 (CLSI, 2008), found that seventeen isolates showed decreased susceptibility to fluconazole. Mutation screening was performed by the amplification of four regions of the ERG11 gene with specific primers designed in this study for each Candida species, followed by electrophoresis SSCP analysis and sequencing. We identified 217 mutations, including 185 silent mutations and 32 missense mutations. Missense mutations were observed in 19 isolates and 17 distinct residues, 7 of them still not described earlier: L321F in C. albicans, K53M in C. krusei; Y221F, K344T, V362M and R371S in C. tropicalis, and R398I in C. parapsilosis. Comparing the results between SSCP electrophoresis technique and sequencing, there was no direct association between changes in electrophoretic migration and changes in the nucleotide sequence of the ERG11 gene. We suggest that the high frequency of changes in ERG11 gene of Candida should be considered in the design of new drugs targeting the enzyme ERG11p

<p>  </p> <p>Fungal pathogens are an increasing threat to humans, plants, and animals worldwide. Death and disease caused by fungal pathogens results in the loss of over 1.5 million lives, 12 million tons of crops, and even entire species every year. <em>Candida </em>species are the leading cause of invasive fungal species lead by <em>Candida albicans, </em>and <em>Candida glabrata </em>in second. <em>Candida glabrata </em>intrinsically has a low susceptibility to azole treatment, and multidrug resistant isolates are becoming more common. Additionally, new emerging <em>Candida </em>species have been found, and most clinical isolates are resistant to one or more drugs. There is a critical need to better understand drug resistance and pathogenesis to generate new therapies. </p> <p>Drug resistance can be caused by several different genetic factors, but until recently epigenetic factors have been frequently overlooked. Epigenetic research has revolutionized the treatment and detection of many cancers. And now, early research has shown epigenetic mechanisms play a role in drug resistance and pathogenesis in fungal species. Limited resources exist to combat fungal infections and understanding the epigenetic mechanisms that contribute to drug resistance and pathogenicity will provide new drug targets for future treatment.</p> <p>Previous publications from the Briggs’ lab showed Set1-mediated histone H3K4 methylation was necessary for proper ergosterol homeostasis and Brefeldin A resistance. One of the three classes of antifungals, azoles, target the ergosterol pathway. The ergosterol connection resulted into this thesis project, investigating the role of Set1-mediated histone H3K4 methylation in drug resistance and pathogenicity in <em>Saccharomyces cerevisiae, Candida glabrata, Candida albicans, </em>and <em>Candida auris. </em>This research was the first to characterize the Set1 complex in <em>C. glabrata </em>and show it is the sole histone H3K4 methyltransferase in <em>C. glabrata </em>and <em>C. auris. </em>Additionally, it shows loss of <em>SET1 </em>in <em>C. glabrata </em>and <em>C. auris </em>reduces pathogenicity and alters drug efficacy. Interestingly, although the loss of <em>SET1</em> seems to cause a similar pathogenic defect in all three <em>Candida </em>species, the role Set1 plays in drug efficacy including which drug and severity varies amongst species and isolates. Altogether, this research project provides new possible drug targets for fungal treatment and knowledge added to the scientific community on the role of epigenetics in fungal pathogens. </p>